H6 



'Wis.—lO 



INFLUENCE OF NITRATES ON NITROGEN- 
ASSIMILATING BACTERLV 



BY 



T. L. HILLS 



IT ^.V!* 



'lO 



Reprinted from JOURNAL OF AGRICULTURAL RESEARCH 

Vol. XII, No. 4 : : : Washington, D. C., January 28, 1918 




PUBLISHED BY AUTHORITY OF THE SECRETARY OF AGRICULTURE. WITH THE COOPERATION 
OF THE ASSOCIATION OF AMERICAN AGRICULTURAL COLLEGES AND EXPERIMENT STATIONS 



WASHINGTON : GOVERNMENT PRINTING OFFICE : 1918 






INFLUENCE OF NITRATES ON NITROGEN-ASSIMILAT- 
ING BACTERLV 

By T. L. Hills, - 
Research Bacteriologist, Idaho Agricultural Experiment Station 

INTRODUCTION 

RElvATlON OF NITRATES TO VARIOUS FORMS OF PLANT LIFE 

The importance of nitrogen to plant life can not be overestimated. 
It is one of several elements essential to plant growth, one, moreover, 
which is apt to be deficient in arable soils. These facts are well brought 
out by the almost innumerable investigations which have been made 
concerning the source of nitrogen for plants. 

The influence of nitrate nitrogen on various plants has been the con- 
trolling idea in many of these experiments. Very little attention has 
been placed on the effect of nitrates on the lower plants, especially the 
bacteria. Because of the relation that exists between higher plants and 
bacteria it seems advisable to consider the effect of nitrates on the soil 
bacteria. Indeed, progress in the knowledge of nitrogenous fertilizers 
depends on a study of the effect of the fertilizer on the soil organisms as 
well as on the higher plants. The action of fertilizers on the different 
groups of soil organisms, the relation of these organisms to higher plants, 
and the separation of the important from the unimportant groups are 
some of the factors involved in the problem of soil fertility. 

REVIEW OF LITERATURE 

The relation of nitrates to the germination of seeds has been studied 
by De Chalmot (Ix)^ who found that corn germinated in solutions con- 
taining nitrate was more robust than corn germinated under similiar 
conditions without nitrate. He also noted that if too concentrated 
solutions of nitrate were used germination was retarded rather than 
hastened. The presence of nitrate also increased the amount of al- 
buminous material in the seed. 

The direct influence of nitrate nitrogen on the growing plant is too 
well known to justify any lengthy discussion here. Jost (26, p. 134) 
gives the results of experiments made by Boussingault, who grew the 
sunflower (Helianthus argophyilus') in sand with and without nitrate. 



'Major portion ot a paper submitted in partial fulfillment of the requirements for the degree of doctor of 
philosophy in bacteriology in the Graduate School of the University of Wisconsin, December. 1916. 

2 The writer wishes to acknowledge his appreciation oi the suEgestions and criticisms obtained through- 
out the progress ol this work from Prof. E. B. Fred and E. G. Hastmgs, of the University of Wisconsin. 

3 Reference is made by number (italic) to "Literature cited," pp. 227-230. 



Journal of Agricultural Research, Vol, XII, No. 4 

Washington, D. C. J*n. 28. 1918 

Ir Key No. Wis. — 

(183) 



i84 



Journal of Agricultural Research 



Vol. XII, No. 4 



During the three months' growth of the plants 1.40 gm. of potassium 
nitrate were added. At the end of the period the dry weight of the 
plant suppHed with nitrate was nearly 60 times greater than that of the 
plant where no nitrate was added. The relation between the growth of 
nonleguminous plants and the amount of nitrate nitrogen supplied is 
shown in a very striking manner in the following table taken from Hell- 
riegel and Wilfarth {21, p. 53-54) : 



Nitrogen as Ca (N03)8 added to pots, 
gm 



Dry weight of oats (grain and straw) . 
gin. . 



None 



o. 3605 
.4191 



0.056 

5- 9024 
5-8510 
5- 2867 



10. 9814 
10. 9413 



0.168 



21. 2732 
21. 4409 



0-336 



But little work has been done on the direct influence of nitrates on the 
development of the Eumycetes. Some investigations have been made as 
to the ability of certain fungi to assimilate nitrate nitrogen directly. 
Ritter {42) studied many species and found that some forms would 
assimilate nitrate directly, while others reduced it to nitrite and am- 
monia. He found some forms which failed to grow on media containing 
nitrate. Kossowicz {28) found that various fungi utilized nitrates and 
that nitrite and ammonia were produced. 

Miinter {36) studied the influence of inorganic salts on the growth of 
various Actinomycetes. He found that potassium and sodium nitrates 
in quantities equivalent to 5 per cent permitted good growth of the 
organisms 'but retarded spore formation. Calcium, barium, and stron- 
tium nitrates in small quantities affected some species but not others. 
Small quantities of these nitrates did not affect growth to any extent, 
but larger quantities were detrimental to growth and spore formation. 
Silver nitrate in all amounts studied almost entirely prohibited growth. 

Nitrates appear to exert some influence on the yeasts. Drabble and 
Scott {13) studied the effect of sodium nitrate on these organisms. They 
found that the greatest reproduction took place in solutions containing 
0.2 gram-molecule of the nitrate. Increasing amounts of the salt led 
to a decrease in reproductive activity until with 0.7 gram-molecule 
present no reproduction took place. From their results it is evident 
that small quantities of nitrate stimulated reproduction, whereas larger 
amounts proved detrimental. Kayser (27) studied the effect of man- 
ganese nitrate on yeasts. He found that the amount which produced 
the maximum increase in the alcoholic fermentation of sugar varied with 
the strain of yeast employed. He likewise found that manganese nitrate 
produced greater increase than did the same quantity of potassium ni- 
trate. Fernbach and Lanzenberg {14) concluded that nitrates hindered 
the rapidity of cell multiplication of yeasts but greatly accelerated the 
action of the zymase. More alcohol was formed in the presence than in 
the absence of nitrate. According to Kossowicz {28), nitrates are not a 
suitable source of nitrogen for yeasts. 



•k"^* 



Jan. 28, i9i8 NitYogen-Assimilating BacteHa 185 

The direct influence of nitrates on bacteria has been studied to a limited 
extent. The influence of various nitrates on soil bacteria has been 
studied by Greaves (jp). He added sodium, potassium, calcium, mag- 
nesium, manganous and ferric nitrates to soil in varying quantities. 
The amount added to the soil was such that in each case equivalent 
quantities of the anion (NO3) in the various forms were added. The 
efiFect of these salts on the bacteria was determined by using ammonifi- 
cation as an index of the bacterial activity. He found that sodium- 
potassium, manganous and ferric nitrates in small amounts, approxi- 
mately 0.97 to 5.5 mgm. of nitrate in 100 gm. of soil, slightly stimulated 
ammonification. Greater concentrations of these salts proved toxic as 
evidenced by a decrease in the amount of ammonia formed. Sodium 
nitrate was much more beneficial to ammonification than potassium 
nitrate. From his results as a whole Greaves concludes that it is the 
electronegative ion which stimulates bacterial activity. Calcium and 
magnesium nitrates proved toxic in all concentrations studied. 

However, a majority of the investigations have been directed toward 
a determination of the effect of the bacteria on the nitrates. But little 
work appears to have been done on the direct action of nitrates on 
bacteria. Pfeffer {38, p. 351) cites some experiments showing the 
repellant action of potassium nitrate toward certain bacteria. Spir- 
illum undula was repelled by a solution of potassium nitrate having an 
osmotic concentration equivalent to 0.5 to i.o per cent. With Spirillum 
volutans a much higher concentration was necessary to bring about the 
same reaction. It was found that different organisms required different 
quantities of the same nitrate to repel them. 

It can be readily seen that by far the greatest amount of work on the 
relation of nitrates to plant growth has been done in the realm of the 
higher plants. Obviously further investigations should be made in 
respect to the effect of nitrates on the lower forms of plant life, especially 
the bacteria. In this paper an attempt is made to set forth the results 
secured in a study of the influence which nitrates exert on certain groups 
of soil bacteria, including not only their reproduction but also some of 
their physiological properties. 

EXPERIMENTAL WORK 

OUTLINE OP PROBLEM 

The results of much careful experimentation show that nitrate nitro- 
gen is most readily assimilated by higher plants. As a rule it seems to 
stimulate the plant to increased activity. In some cases this is un- 
doubtedly due to increased nutrition, while in others it is a result of 
nuclear stimulation with a consequent cell multiplication. No sharp 
line can be drawn between these two effects. Probably one overiaps 
the other, and the increased growth of the organism can be attributed 
to a combination of the two actions. 



1 86 Journal of Agricultural Research voi. xii, no. 4 



From a practical standpoint the relation of nitrates to the nitrogen- 
assimilating organisms of the soil is of importance. Hence, it was 
arranged to study the effect of nitrates on soil bacteria, especially those 
forms concerned with the fixation of atmospheric nitrogen. The work 
naturally falls into two rather distinct lines of investigation. First, the 
influence of nitrates on Azotobacter was determined. Here studies were 
made of the effect of nitrates on the growth of the organism in soil and 
also the effect of these salts on the nitrogen-fixing property of these 
bacteria. The action of Azotobacter on nitrates in solution, the relation 
of nitrates to pigment production and to the formation of volutin bodies 
were studied. Second, the influence of nitrates on the growth of Bacillus 
radicicola in soil was studied. The action of B. radicicola on nitrates in 
solution and the possible nitrogen-assimilating properties of the legume 
in the presence of nitrates were investigated. Also the influence of 
nitrates on gum production was determined. The latter part of the 
investigations included a study of the relation of nitrates to nodule 
formation on alfalfa. 

METHODS USED IN EXPERIMENTS 

Nitrates were determined by the reduction method with Devarda's 
alloy and also by the phenolsulphonic acid (colorimetric) method. 

The total nitrogen content of all samples was determined by the 
modified Kjeldahl method with sulphuric acid, salycilic acid, sodium 
thiosulphate, and copper sulphate. Where nitrate nitrogen was present, 
50 c. c. of concentrated sulphuric-sal ycilic acid (25 c. c. of concentrated 
acid plus 25 c. c. of distilled water) were added to the cultures slowly 
and with constant stirring. This acid was allowed to react for a few 
days, after which the usual procedure was carried out. Digestion was 
continued for five to six hours subsequent to the clarification of the 
liquid. 

The amount of ammonia was determined by distillation with steam 
in the presence of magnesium oxid. 

Nitrites (qualitative test) were tested for with Trommsdorf's reagent. 

In all distillations NI14. acid and alkali were used. 

In the preparation of agar cultures of alfalfa seedlings the seeds were 
treated with a 0.25 per cent solution of mercuric chlorid and rinsed in 
sterile distilled water. Three bacteria-free seeds were transferred to the 
surface of soft mannit agar (0.7 per cent agar) in each tube. 

The nitrates were added in solution to all cultures. Gram-molecular 
quantities of potassium, sodium, calcium, and ammoniun nitrates 
(Merck's) were weighed into sterile distilled water. These solutions 
were prepared in such a manner that 5 c. c. contained 450 mgm. of 
nitrate. In all nitrate solutions the nitrate radical, or anion, was 
present in the same quantities, while the cation, or metal, was present 
in varying quantities, depending upon the particular salt. 



Jan. 28. i9i8 Nitrogen-Assimilating Bacteria 187 



Plate counts of all soil cultures were made by weighing 20 gm. (dry 
weight) of the soil into a 200-c. c. water blank. From this suspension 
all subsequent dilutions were made. Mannit agar ^ was used for the 
plate counts in the cultures of Azotobacter and B. radicicola. Duplicate 
plates were made for each dilution poured. 



SOIL USED 



Only one type of soil was employed, Miami silt loam obtained from 
the Experiment Station farm. No chemical analyses of the soil were 
made other than an estimation of its organic matter content, which was 
approximately 2.75 per cent. The soil was neutral in reaction and its 
nitrate content was approximately 1.5 mgm. of nitrogen as nitrate in 
100 gm. of the dry soil. 

ISOLATION OF AZOTOBACTER AND BACILLUS RADICICOLA 

Azotobacter. — (i) Strain A was isolated from a silt loam soil. This 
strain grew well on mannit agar, but produced no pigment after three 
weeks' growth. (2) Strain B was isolated from a sandy loam soil. 
This strain grew equally well on mannit agar and produced a brownish 
black pigment within one week's growth. Both strains assimilated 
practically the same amount of atmospheric nitrogen under laboratory 

conditions. 

Bacillus radicicola.— A stock laboratory culture of B. radicicola 
was replated twice before taking the final culture. The nodule produc- 
ing power of the organism was determined by inoculating bacteria-free 
alfalfa seedlings (in soft agar). After sufficient incubation nodules were 
produced in abundance. 

INFLUENCE OE NITRATES ON AZOTOBACTER 

INFLUENCE OF NITRATES ON THE GROWTH AND REPRODUCTION OF AZOTOBACTER IN 

STERILIZED SOIL 

What effect do nitrates have on pure cultures of Azotobacter in ster- 
ilized soil? Do these salts cause a decrease in the numbers of the organ- 
isms? Do they cause an increase in numbers? Or do they exert no 
particular influence one way or the other? It is difficult to believe that 
the latter could be true, inasmuch as nitrates have such a profound 
effect on higher forms of plant life. Such readily soluble and assimilable 
substances as nitrates could hardly remain without affecting either an 
increase or a decrease in the number of organisms existing in their 

presence. 

With the idea of determining what effect nitrates might have on 
Azotobacter when grown in sterilized soil, the following experiments were 
planned. In this work both strains of the Azotobacter (described on 

1FRED.E.B. A I^BORATORYMANUAl, OF SOIL BACTERIOLOGY, p. io8. Philadelphia and London, 1916. 



i88 



Journal of Agricultural Research 



Vol. XII, No. 4 



p. 187) were employed and conditions governing the preparation and 
incubation of the cultures were similar in the case of each strain. The 
only variation was the periods used in incubating the cultures. Counts 
were made after one and two weeks' incubation with strain A and after 
one, two, and three weeks with strain B. 

TABiyE I. — Influence of potassium nitrate on the growth of Azotobacter (strain A) in 

sterilized soil 





Treatment 

(nitrate 

in 100 

gm. of 

dry soil). 


Number of organisms in i gm. of drj- soil. 


Culture No. 


At begin- 
ning. 


After I week. 


Relative. 


After 2 weeks. 


Relative. 


I 


Mgm. 





10 

10 

25 

25 

50 

50 

100 

100 

150 

150 

200 

200 

300 

300 


15, 600 
15, 600 
15, 600 
15, 600 
15, 600 
15, 600 
15,600 
15, 600 
15, 600 
15, 600 
15,000 
15, 600 
15, 600 
15, 600 
15, 600 
15, 600 


825, 000 

935. 000 

I, 500, 000 


Per cent. 
> 100 

} 170 

} 523 

} 2, 233 

} 1.295 
} 179 
} 27 

} " 


f 315,000 

L 360, 000 
f 1, 175, 000 


Per cent. 


2 


> 100 


■7 






} 348 


t 


4, 200, 000 
5, 000, 000 
20, 400, 000 
18, 900, 000 
11,000,000 
II, 820,000 


1' 12, 350, 000 
t 10, 750, 000 

r 27, 750, 000 




6 


} 3.418 


7 




8 


1 8,210 


Q 


f 9, 000, 000 
t 9, 150, 000 
f 25, 000 
[ 55, 000 
r 
I 

{ c 




10 


} 2, 68s 






12 


I, 575. 000 
225, 000 
250, 000 





1 ^^ 


I^ 




TA 


( ° 


je 




16 


1 ° 







Table II. — Infltience of sodium nitrate on the growth of Azotobacter {strain A) in 

sterilized soil 





Treatment 
(nitrate 
in 100 
gm. of 

dry soil). 


Number of organisms in i gm. of dry soil. 


Culture No. 


At begin- 
ning. 


After I week. 


Relative. 


After 2 weeks. 


Relative. 


I. 


Mgm. 



10 

10 

25 

25 

SO 

SO 

100 

100 

150 

150 

200 

200 

300 

300 


13,800 
13, 800 
13, 800 
13, 800 
13, 800 
13, 800 
13, 800 
13, 800 
13, 800 
13, 800 
13, 800 
13, 800 
13, 800 
13, 800 
13, 800 
13, 800 


310, 000 
225, 000 

575. 000 

430, 000 

2, 850, 000 

5, 800, 000 

15, 200,000 
12, 750, 000 
17.750,000 

16, 200, 000 
550, 000 
400, 000 







Per cent. 
\ 100 

1 188 
f I. 615 
} 5.217 
} 6,335 
} 177 
1 ° 
1 ° 


r 425, 000 
\ 490, 000 
f 875, 000 


Per cent. 


2 


t 100 
} 191 
} 492 

} 3, 150 
1 2, 800 


2 


4 


e 


/ 2, 250, 000 


6 


7 , 


f IS, soo, 000 
I i3> 300, 000 
f 9, 850, 000 
I 15.750,000 
f 690, 000 
I 375. 000 

r . 
I 
r 
\ 


8 


Q 


10 


II 


} - 
} ° 
} ° 


12 . 


XX 


14 


IC 


16 





Jan. 38. 1918 



Nitrogen-A ssimilating Bacteria 



189 



Table III. — Influence of calcium nitrate on the growth of Azotobacter {strain A) in 

sterilized soil 





Treatment 

(nitrate 

in 100 

gm. of 

dry soil). 


Number of organisms in i gm. of dry soil. 


Culture No. 


A,t begin- 
ning. 


After I week. 


Relative. 


After 2 weeks. 


Relative. 


I 


Mgm. 





10 

10 

25 

25 

50 

50 

100 

100 

150 

150 

200 

200 

300 

300 


p p p pppppppppppp p 
OOOOOOOOOOOOOOOO 


260, 000 

330, 000 

5, 800, 000 


Per cent. 
\ 100 

1 1, 966 
1 3.440 


\ 3 10, 000 
1 260, 000 

1 975. 000 
[ 1, 090, 000 
/ 9, 200, 000 
\ 8, 600, 000 

r 13, 200, 000 
\ 12, 600, 000 

/ 8, 750, 000 
\ 8, 000, 000 
f 2,000,000 
I 2,350,000 

/ ° 

r 
I ° 


Per cent. 


2 


\ 100 


•2 


I 362 




e 


10, 700, 000 

9, 600, 000 

i^, 2t;o, 000 




6 


\ 3. 122 


7 


1 

} 4, 526 


8 


11,600,000 f 4,213 1 




6, 600, 000 
6, 050, 000 
3, 500, 000 
3, 900, 000 




I 2, 144 
} 1.254 

1 ° 


} 2,938 


10 




I 763 


12 


1-2 




14, 


1 ° 


le 


ll 




16 


1 ° 






1 





One hundred and fifty gm. of soil (dry weight) were weighed into 500- 
c. c, Erlenmeyer flasks and the nitrates added in solution, as indicated in 
the following tables. At the same time i per cent of mannit was added 
in solution and the moisture content was raised to approximately 18 per 
cent. The flasks were allowed to remain at room temperature for one 
day, when the contents were thoroughly mixed. The flasks and contents 
were then sterilized at 15 pounds' pressure for three hours. Upon cooling 
they were inoculated with 5 c. c. of a suspension of the organisms in sterile 
distilled water. The cultures were incubated at 28° C. and counts made 
at the intervals already indicated, Mannit agar was used in pouring the 
plates. Each number in the following tables represents an average of 
duplicate plates. Tables I, II, and III show the results of the work with 
strain A and Tables V, VI, and VII the results with strain B. 

It will be seen at a glance that all three nitrates exerted an enormous 
influence on the growth of the Azotobacter. The smallest concentration 
did not appear to exert much influence either in increasing or decreasing 
the number of Azotobacter. There was a slight gain, but it was not so 
marked as that brought about by higher concentrations of nitrates. 
When 25, 50, and 100 mgm. of nitrate were present in 100 gm. of soil, 
very large increases were obtained in practically all instances. In one 
instance sodium nitrate caused the greatest relative gain, but the most 
consistent increase was produced by calcium nitrate. Beginning with 
150 mgm, the number of Azotobacter began to decrease. This decrease 
was especially noticeable in the cultures containing potassium and 
sodium nitrates. At the end of the first week, Azotobacter organisms 



IQO 



Journal of Agricultural Research 



Vol. XII, No. 4 



were still found in the potassium-nitrate cultures where 200 mgm. were 
present. However, at the end of the second week the organisms were 
dead. The same concentration of sodium and calcium nitrates proved 
even more toxic. No evidences were secured, indicating that these 
organisms can resist concentrations in excess of 300 mgm. of nitrate per 
100 gm. of soil. 

The question may be raised in regard to the influence of sterilization 
on the nitrate present in the soil, Does the prolonged heating in the 
presence of soil organic matter reduce the nitrate? In order to study 
this point, a few cultures were prepared similar to those already described. 
They were subjected to sterilization under pressure of 15 pounds for two, 
three, and five hours. Nitrate determinations at the end of these periods 
failed to show any reduction. In the presence of i per cent of mannit 
the nitrate content remained unchanged during sterilization. 

From these results it is evident that small amounts of nitrate up to 
150 mgm. of nitrate in 100 gm. of soil greatly increased the reproduction 
of x\zotobacter. In regard to the toxicity of higher concentrations, 
sodium nitrate appeared to exert the greatest influence in this direction, 
followed by calcium and potassium nitrates in the order named. The 
results of the experiment are recorded in Table IV. 

Table IV. — Influence of ammonium nitrate on the growth of Azotohacter {strain A) in 

sterilized soil 





Treatment 

(nitrate 

in 100 

gm. 01 

dry soil). 


Number of organisms in i gm. of dry soil. 


Culture No. 


At begin- 
ning. 


After I week. 


Relative. 


After 2 weeks. 


Relative. 


I 


Mgm, 





25 

25 

100 

100 

200 

200 


18, 500 
18, 500 
18, 500 
18, 500 
18, 500 
18, 500 
18, 500 
18, 500 


I, 400, 000 

1, 050, 000 
5, 600, 000 
4, 900, 000 

2, 900, 000 
2, 600, 000 
1, 100, 000 

950, 000 


Per cent. 
> 100 

; 427 
} 223 
} 84 


r 975, 000 
\ 1, 100, 000 
f 5, 000, 000 
\ 3, 900, 000 
/ 3. 950, 000 
\ 4, 100,000 
f 875, 000 

I 915) 000 


Per cent. 
> 100 


2. .. . . 


■J 


4 


e. 


6 


} 388 
1 86 


7. .......... . 


8.... 





That the nitrate radical and not the combined metal was the causal 
agent in the increase in the number of Azotobacter was indicated from 
the results of the next test. Here ammonium nitrate was used. 

It will be seen from the data of this experiment that ammonium nitrate 
caused an increase in the number of Azotobacter when present in small 
amounts. However, the increase in the presence of ammonium nitrate 
was less marked than when equal quantities of the other nitrates were 
used. Since the experiments with ammonium nitrate were not made at 
the same time as the preceding experiments (discussed on pp. 189-190), it 
is possible that conditions varied sufficiently to account for the less pro- 
nounced results. When 200 mgm. of nitrate were present in loo gm. of 



Jan. 38, X918 



Nitrogen- A ssimilating Bacteria 



191 



soil the number of Azotobacter showed a decrease. Apparently ammo- 
nium nitrate is more toxic than potassium, sodium, and calcium nitrate. 
However, the main point at issue seems fairly well established — namely, 
that the increase in the number of Azotobacter is caused by the nitrate 
radical and not by the combined metal. 



Table V. — Influence of potassium nitrate on the growth of Azotobacter (strain B) in 

sterilized soil 





Treat- 
ment 

(nitrate 
in 

100 gm. 
of dry 
soil). 


Number of organisms in i gm. of dry soil. 


Cultu 
No. 


At begin- 
ning. 


After I week. 


Relative. 


After 2 weeks. 


Relative. 


After 3 weeks. 


Rela- 
tive. 


I. . 

2 . . 


Mgm. 


10 
10 
25 
25 
50 

50 
100 

100 
150 
150 
200 
200 
300 
300 


12, 600 
12, 600 
12, 600 

12, 600 
12, 600 
12, 600 
12, 600 
12, 600 
12, 600 
12, 600 
12, 600 
12, 600 
12, 600 
12, 600 
12, 600 
12, 600 


235, 000 


Per cent. 
\ 100 

>I, 510 

}2, 436 

|i.340 

|l,320 

} 851 
r 373 
f ° 


/ 112,500 
\ no, 500 
/ 2, 100, 000 
\ 2, 250, 000 
/ 1,575.000 
\ 1,950,000 

r 3,250,000 
\ 4, 900, 000 
{ 4, 000, 000 
\ 3, 300, 000 
f 2, 000, 000 
\ 2, 100, 000 
{ 800, 000 
1 750, 000 

{ 


Per cent. 

> 100 

}i,9So 
}i,58r 
J3, 65s 
]i, 3(>l 
}i.838 
} 695 
1 ° 


f 116,000 
\ 117,000 
{ 875, 000 
\ I, 260, 000 
/ I, 700, 000 
'1 1,325,000 
f 3,525,000 

\ 2, 960, 000 
f 2, 500, 000 

\ 2, 900, 000 
f I, 500, 000 

\ 2, 000, 000 
f 630, 000 

\ 700, 000 

{ I 


Per ct. 
\ 100 

}i,300 
}2, 783 
}2,3i7 
\i, 502 

} 580 

} ° 


3- ■ 

4- ■ 

5- ■ 
6. . 

!■■ 
8.. 
9.. 

10. . 

11 . . 

12. . 

13- • 
14. . 

15- • 
16. . 


3, 750, 000 
3, 300, 000 

5, 750, 000 

5, 700, 000 

3, 100, 000 

3, 200, 000 

3, 200, 000 

3, 000, 000 

2, 100, 000 

I, 900, 000 

875, 000 

880, 000 







Table VI. — Influence of sodium nitrate on the growth of Azotobacter {strain B) in 

sterilized soil 





Treat- 
ment 

(nitrate 
in 

100 gm. 

of dry 
soU). 


Number of organisms in i gm. of dry soil. 


Cultu 

No. 


At begin- 
ning. 


After I week. 


Relative. 


After 2 weeks. 


Relative. 


After 3 weeks. 


Rela- 
tive. 


1. . 

2. . 

3-- 
4- • 
5-- 
6. . 

7-- 
8.. 
9.. 

10. . 

11 . . 

12. . 

13- ■ 
14.. . 


Mgm. 





10 

10 

25 

25 

50 

50 

100 

100 

150 

150 

200 

200 

300 

. 300 


15, 600 
15, 600 
15, 600 
15,600 
15,600 
15, 600 
15,600 
15,600 
15, 600 
15, 600 
15, 600 
15, 600 
15,600 
15,600 
15,600 
15,600 


158, 000 
149, 000 

I, 250, 000 
990, 000 

I, 765, 000 

1, 825, 000 
1,875,000 

2, 250, 000 
2, 200, 000 
I, 950, 000 

165, 000 
170, 000 






Per cent. 
> 100 

} 727 
}l, 165 
}i,338 

}i,35o 
1 108 

} ° 
1 ° 


f 110,500 
\ 126, 000 
f I, 750, 000 
\ 1,350,000 
f 6, 600, 000 
\ 5, 300, 000 
f 2, 025, 000 
\ 3, 040, 000 
f 2,775,000 
\ 3, 200, 000 
f 530, 000 
L 785, 000 

{ I 
{ I 


Per cent. 
> 100 

[1,310 

}5,o29 

>2, 141 

}2,525 

} 556 






/ 112,500 
I 115,000 
f 5,000,000 
\ 6, 600, 000 
1 9, 150, 000 
\ 7, 150, 000 
ri5,95o,ooo 
\i4, 600, 000 
f 5, 800, 000 
1 5, 250, 000 
1 3, 100, 000 
\ 2, 750, 000 

/ ° 
1 

{ 


Per cl. 
\ 100 

} 5,097 

\ 7, 161 
}i3, 423 
1 4,860 
} 2, 573 
I ° 
1 ° 



192 



Journal of Agricultural Research 



Vol. XII, No. 4 



Table VII. — Influence of calcium nitrate on the growth of Azotohacter {strain B) in 

sterilized soil 



Culture 
No. 



Treat- 
ment 
(nitrate 

in 
100 gm 
of dry 
soil). 



Mgm. 

o 

o 

10 

10 

25 
25 
50 
50 

100 
100 

150 
150 

200 
200 

300 
300 



Number of organisms in i gm. of dry soil. 



At begin- 
ning. 



22, 000 
22, 000 
22, 000 
22, 000 
22, 000 
22, 000 
22, 000 
22, 000 
22, 000 
22, 000 
000 
1, 000 
000 
000 
000 
000 



22 



After I week. 



905> 
860, 
23, 200, 
19, 600, 
17, 200, 
19, 600, 
II, 800, 
14, 000, 

7, 500. 
II, 000, 

2, 55o» 

3. 500. 
107, 

87, 



000 
000 
000 
000 
000 
000 
000 
000 
000 
000 
000 
000 
500 
500 



Relative. 



Per cent. 
\ 100 

y, 423 

>2, 084 
|l, 461 

} 342 



After 2 weeks. 



475, 000 
460, 000 
28, 000, 000 
36, 000, 000 
52, 000, 000 
43, 500, 000 
22, 500, 000 
20, 000, 000 
12, 000, 000 



5, 300, 000 

6, 500, 000 

2, 750, 000 

3, 225, 000 



Relative. 



Per cent. 
> 100 

W 181 

]z, 25s 

jl, 448 

I 818 

402 

203 



After 3 weeks. 



130, 000 

157, 500 
050, 000 
600, 000 
750, 000 
250, 000 
400, 000 
850, 000 
750, 000 
950, 000 
800, 000 



130, 000 

120, 000 

o 



Rela- 
tive. 



Per ct. 
\ 100 

|3, 002 

}2-> 273 

J2, 633 

}i,7& 



o 
420 
II 



A glance at the figures of Tables V, VI, and VII shows that the small- 
est concentration of nitrate used produced a much more marked relative 
increase in numbers with strain B than it did with strain A. On the 
other hand, the greater resistance of this strain to the higher nitrate 
concentrations is clearly evident. In the potassium- and calcium- 
nitrate cultures the organisms were present in an active state where 
the nitrate was added in amounts equivalent to 200 mgm. of nitrate 
in 100 gm. of soil. However, this same concentration of sodium nitrate 
prevented the development of the Azotobacter. The first five concen- 
trations of all three nitrates caused a very large increase in the number 
of Azotobacter when compared with control cultures where no nitrate 
was added. In one instance an enormous increase was noted after three 
weeks' incubation in the presence of 50 mgm. of nitrate as sodium nitrate. 
This increase far excelled that noted with other concentrations of the 
same salt. The writer can ofifer no conjecture as to this occurrence. 

Similar results were obtained by the writer in 19 14 (23) with a strain 
of Azotobacter isolated -from a silt loam soil at the Pennsylvania Experi- 
ment Station. It was found that soil and liquid cultures containing 
small amounts of potassium, sodium, and calcium nitrates caused an 
increase in the number of Azotobacter in pure culture compared with 
control cultures containing no nitrate. An increasing concentration of 
the nitrates continued favorable to the growth of the organism up to a 
certain limit, but higher concentrations retarded its growth. Finally 
a nitrate concentration was attained at which Azotobacter growth 
altogether ceased. 



Jan. 28, igis NUrogen-Assimilating Bactcria 193 

The results of the study of nitrates and their influence on Azotobacter 
in sterilized soil show very clearly that small amounts of nitrate cause 
a great increase in the number of Azotobacter cells. Higher concentra- 
tions are not so favorable to the growth of the organisms, and the highest 
concentrations studied prevented the development of the Azotobacter 
in sterilized soil. 

From a study of the results of these experiments, it seems that the 
increase in number of Azotobacter in the presence of small amounts 
of nitrate is a direct result of nuclear stimulation. Later studies to 
be cited {pp. 205-208) show that nitrates exerted considerable influence 
on the internal structure of the Azotobacter cell. It appears reasonable 
to expect that the nitrate affected the nuclear structure in such a manner 
that an increase in cell multiplication resulted. It seems probable that 
the action of nitrate as a simple nutrient would be shown by a slower 
increase in cell multiplication. 

INFLUENCE OF NITRATES ON THE FIXATION OF NITROGEN BY AZOTOBACTER 

It has been shown in the preceding paragraphs that the presence of 
small quantities of nitrate in sterilized soil bring about a large increase 
in the number of Azotobacter. This increase was noted in the case 
of both strains of Azotobacter. It would be of interest to know whether 
the increase in bacterial numbers was accompanied by a corresponding 
increase in the amount of nitrogen assimilated. 

The results secured by a few investigators indicate that in the presence 
of combined nitrogen as nitrates the nonsymbiotic nitrogen-fixing 
organisms will not fix atmospheric nitrogen. Stoklasa (44, p. 492-503) 
studied the influence of Azotobacter on sodium nitrate in aerobic and 
anaerobic liquid cultures. He found only a small gain in organic nitro- 
gen and from these results he concluded that in the presence of nitrates 
Azotobacter could not assimilate atmospheric nitrogen. It has been 
shown by Hanzawa {20) that in a liquid culture containing 12 mgm. 
of nitrate (from potassium nitrate) in 100 c. c. of medium, a mixed 
culture of Azotobacter fixed 5.25 mgm. of nitrogen. Under the same 
conditions with 60 mgm. of nitrate present in 100 c. c. of medium he 
found but 5.35 mgm. of nitrogen fixed. He concluded that nitrates 
remained, as far as small quantities were concerned, almost without 
influence on the amount of atmospheric nitrogen fixed by Azotobacter. 

Some studies have been carried on with respect to the influence of ni- 
trates on the nonsymbiotic anaerobic nitrogen-assimilating organism, 
Clostridium spp. Bredemann (9) showed that ammonium nitrate in 
solution caused a decrease in the amount of nitrogen fixed by species 
of Clostridium. Pringsheim (40) grew cultures of C. americanum in 
solutions containing potassium nitrate. He found that in the presence 
of available energy the organism fixed some nitrogen when nitrate was 



194 



Journal of Agricultural Research voi. xii, No. 4 



present but to a less extent than did control cultures containing no 
nitrate. 

From these results it appears that nitrates do not stimulate the nitro- 
gen-assimilation of the nonsymbiotic nitrogen-fixing bacteria. 

Inasmuch as nitrates in small amounts caused such an increase in 
the number of Azotobacter in sterilized soil, it was thought advisable 
to determine just what influence these salts exert on nitrogen fixation 
by Azotobacter. Accordingly, experiments were carried out with 
Azotobacter on agar films, in soil cultures and in solution. 

Agar-film cultures. — In this work both strains of the Azotobacter 
were used. One hundred c. c. of mannit agar were placed in liter 
Erlenmeyer flasks and nitrates of potassium, sodium and calcium 
added in varying quantities. The flasks and contents were sterilized 
at 10 pounds' pressure for 25 minutes, cooled, and inoculated with 10 
c. c. of a suspension of the organism in sterile distilled water. The 
flasks were incubated at 28° C. for three weeks. The weight of both 
inoculated and uninoculated flasks was maintained throughout the 
experiment by the addition of sterile distilled water. At the end of the 
incubation period total nitrogen analyses were made. Because of the 
high nitrate content dilute sulphuric-salycilic acid was added slowly 
and carefully to prevent loss of nitrogen by the evolution of gaseous 
oxids of nitrogen. The acid was allowed to react for a few days before 
continuing the total nitrogen determination. The results of the experi- 
ments are presented in Tables VIII and IX. 

Table VIII. — Influence of nitrates on the fixation of nitrogen by Azotobacter {strain A) 

on agar films 



Cul- 
ture 
No. 



Treatment (nitrate in looc. c. of medium). 



o 

50 mgm. of NO3 potassium nitrate . 

. . . .do 

100 mgm. of NO3 potassium nitrate 

. . . .do 

50 mgm. of NO3 sodium nitrate. . . 

. . . .do 

100 mgm. of NO3 sodium nitrate. 

50 mgm. of NO3 calcium nitrate. . 

....do 

100 mgm. of NO3 calcittm nitrate . 
....do 



Nitrogen contained in loo c. c. of medium. 



Inoculated. 



Found. Average, 



Mgm. 
13.00 
12. 70 

12. 60 
18.50 
18. 40 
27. 60 

27-75 
18.65 
18.30 
27. 00 
27.65 

13-75 

13. 70 
18.80 
19-15 



Mgm. 
12.80 

18.45 
70 



Uninoculated. 



Found. Average. 



Mq'^). 
4- •■> 
4. y 
4. I 
7. oy 

7. 20 
16.80 
15-70 

7- 50 

7-30 

15. 00 

i_5. 20 

8.00 

8. 50 
14. 50 
14.30 



Mgm.. 
4-05 

I 7- 10 
} 16. 25 

} 8-25 
\ 14-40 



Nitrogen 
fixed. 



Mgm. 
8.75 

"•35 
11-45 
II. 10 
12.25 

5-50 
4-55 



Jan. 28, 1918 



Nitrogen- A ssimilating Bacteria 



195 



Table IX. — Influence of nitrates on the fixation of nitrogen by Azoiobacter {strain B) 

on agar films 



Cul- 
ture 
No. 



3 
4 

5 
6 

7 
8 

9 
10 
II 
12 

13 
14 
IS 



Treatment (nitrate in 100 c.c. of medium). 



o 

o 

o 

75 mgm, of NO3 as potassium ni- 
trate 

...do 

150 mgm. of NO3 as potassium ni- 
trate 

....do 

75 mgm. of NO3 as sodium nitrate 

. . . .do 

1 50 mgm, of NO3 as sodium nitrate 

....do 

75 mgm. of NO3 as calcium nitrate 

....do 

1 50 mgm . of NO3 as calcium nitrate 
....do 



Nitrogen contained in 100 c. c. of medium. 



Inoculated. 



Found. Average. 



Mgm. Mgm 



15-50 
15-70 
15.60 

25. 20 
25.40 

36.40 
36.90 
25. 60 
25.70 
37- 60 
37- 20 
20. 10 
19. 60 
32.80 
33-30 



15.60 



25-30 



} 36 
} 37 
\ 33 



Uninoculated. 



Found. I Average. 



Mgm. 

6. 50 
6.30 
6. 40 



\ 14 



{; 



14-70 

24. 00 
23.20 
12.80 
13.20 
26. 20 
25.40 
12. 00 
2. 70 
24-50 
2^. 20 



MgTti. 



6. 40 




Nitrogen 
fixed. 



Mgm: 
9. 20 

11-45 

13-05 
12.65 

II. 60 

7-50 

8. 20 



A glance at the results (Tables VIII and IX) shows that an increase in 
nitrogen fixation occurred where potassium and sodium nitrates were 
present, whereas a marked decrease in the total nitrogen content was 
observ'ed where calcium nitrate was used. Whether the calcium itself 
is detrimental to an increase in organic nitrogen or whether it is the com- 
bination of calcium with nitrate can not be stated. It is significant, 
however, that this decrease in fixation of nitrogen was noted throughout 
all the experiments where calcium nitrate was employed. It is very 
evident that calcium nitrate exerts some detrimental effect on the nitro- 
gen assimilating properties of the organism. 

There seems to be but a slight difference in the nitrogen-fixing ability 
of the two strains studied. In the absence of nitrates the amount fixed 
varies but little. Also in the presence of potassium and sodium nitrates 
the relative increase in amount of nitrogen fixed remains about the 
same. Calcium nitrate offers an exception where it is employed. The 
detrimental efifect seems to be more marked in the case of strain A than 
with strain B. Strain A under normal conditions fixed slightly less nitro- 
gen than strain B, so it may be possible that this strain is weaker. 

The formation of pigment by the Azotobacter in the presence of the 
nitrates is of interest. Strain A normally produced no pigment by the 
end of three weeks' incubation. But when grown on the agar films in 
the presence of nitrate a most marked pigment production appeared. 
This pigment was especially noticeable in the presence of the calcium 



196 Journal of Agricultural Research voi. xii, No. 4 

salt. Since strain B normally produces a good pigment, the influence 
of nitrate on this strain was not very marked. The relation of nitrates 
to pigment formation will be taken up later (pp. 203-205). 

From the results of the experiments with agar films containing various 
amounts of nitrate, it seems apparent that potassium and sodium nitrates 
in amounts of 50 and 100 mgm. of nitrate in 100 c. c. of medium cause a 
small increase in the amount of nitrogen fixed. However, this increase 
in fixation is not at all parallel with the increase in number of Azoto- 
bacter caused by nitrates in sterilized soil. 

It may be concluded that an increase in the number of Azotobacter in 
sterilized soil as a result of nitrate stimulation does not mean a corre- 
sponding increase in nitrogen fixation on agar films. 

Soiiv CULTURES. — The conditions obtaining in these experiments were 
strictly comparable with those heretofore cited dealing with the influence 
of nitrates on Azotobacter in sterilized soil (pp. 187-193). 

The fixation of nitrogen was studied in pure culture in sterilized soil 
and in unsterilized soil. One hundred and fifty gm. of soil (dry weight) 
were weighed into i -liter Erlenmeyer flasks, nitrates were added in vary- 
ing amounts from 10 to 200 mgm., and i per cent of mannit was also 
added. Triplicate flasks were prepared for each amount of nitrate studied. 
The moisture content was raised to approximately 18 per cent and the 
flasks allowed to remain at room temperature for one day. The con- 
tents were then thoroughly mixed and a fine crumb structure produced. 
The flask-s for the experiments with pure cultures in sterilized soil were 
immediately steriHzed at 15 pounds' pressure for three hours. After 
cooling, two of each set were inoculated with 5 c. c. of a suspension of 
Azotobacter (strain A) in sterile distilled water. The remaining flask of 
each set was not inoculated, but was incubated at 28° C. with the inocu- 
lated flasks. The moisture lost by evaporation was replaced from time 
to time by the addition of sterile distilled water. At the end of the incu- 
bation period the soil was removed and spread out in thin layers and 
allowed to dry. It was then thoroughly ground in a porcelain-ball mill 
for one hour. At the end of this time all of the soil passed through a 
loo-mesh sieve. 

Soil cultures used in the study of the effect of nitrates on nitrogen 
fixation in unsterilized soil were prepared in a similar manner, except 
that the flasks were not sterilized. Previous to incubation a small 
inoculum of Azotobacter (strain A) was added to insure the presence of 
the nitrogen-fixing organism in the soil cultures. The proper moisture 
content was maintained in the same manner as in the case of the pure 
cultures in sterilized soil and the incubation period was the same for both. 
The results are given in Tables X, XI, XII, and XIII. 



Jan. 28, 1918 



Nitrogen- Assimilating Bacteria 



197 



Table X. 



-Influence of sodium nitrate on the fixation of nitrogen by Azotobacter in 
sterilized soil 





Treatment 
(nitrare in 
100 gm. of 
dry soil). 


Total tiitrogen in 100 gm. of dry soil. 


Nitrogen 


Culture No. 


Inoculated. 


Uninoculated. 


fixed in 
100 gm. of 




Found. 


Average. 


Found. 


Average. 


dry soil. 


I 


Mgm. 













10 

10 

10 

10 

10 

10 

50 

50 

50 

50 

50 

50 

150 

150 

150 

150 

150 

150 


Mgm. 
135-0 
134. 
132. 
133-0 
137-0 


Mgm. 

■ 133- 7 

■ 135-0 
136.6 
137. 
149- 
149. 2 
162. 3 
162. 5 


Mgm. 
132. 

131- 5 

131. 


Mgm. 

\ 131- 5 


Mgm. 


I 


2-7 


I 


2 




2 






3-5 


2 






■J 


137-0 
136.0 
137-0 
136.5 
137.5 
137.0 
149.0 
149.0 
149.0 
148. 5 
149-5 
149-5 
163.0 
162. 
162. 
162. 5 
163.0 
162. 


134. 
133-5 
134- 


^33- 7 




■i 


2.9 


•J 


4 




4 






[ 3-3 


4 






C 


140. 
137-0 

138- 5 


138.5 




C 


10.5 


C 


6 




6 


i 


[ 10.7 


6 






7 

7 


152. 
150.0 
152. 5 


[ 151-5 


10. 8 


7 . , 




8 


1 


8 






> II. 


8 






J 









Table XI. — Influence of sodium nitrate on the fixation of nitrogen by Azotobacter in 

unsterilized soil 





Treat- 
ment (ni- 
trate in 
100 gm. ol 
dry soil). 


Total nitrogen in 100 gm. of dry soil. 


Nitrogen 


Culture No. 


Inoculated. 


Uninoculated. 


fixed in 
100 gm. of 




Found. 


Average. 


Found. 


Average. 


dry soil. 


I 


Mgm. 













10 

10 

10 

10 

10 

10 

50 

50 

50 

50 

50 

50 

150 

150 

150 

150 

150 

150 


Mgm. 
132.0 
135-0 
135-0 
132.0 
134.0 
134.0 

137-5 
138.8 
138.8 
137-5 
137-5 
138.0 

150. 

151. 
150. 
149.0 
149-5 
150. 5 
169. 

167. 
168.0 

167.5 
168.0 

168. s 


Mgm. 
[ 134- 

[ ^33- 3 

137- 8 

1 137- 7 

[ 150- 3 

[ 149- 7 

[ 168. 

i 168. 


Mgm. 
f 130. 

\ ^33- 5 
I 132- 


Mgm. 
131. 8 


Mgm. 


I 


2 2 


I 




2 




2 






I- 5 


2 . . . 






•2 


f 134- 
1 ^33- 
I 133- 


1 ^33- 3 




•3 


4-5 


■3 


A 


1 


A 






4-4 


4 






c 


[ 140. 
\ 140. 5 
t 142. 


\ 140. 8 




c 


9-5 


c 


6 


1 


6 






8.9 


6 






7 


[ 148. 
{ 154- 
I 153- 5 


151. 8 




7 


16. 2 


7 




8 


1 


8 






\ 16. 2 


8 






J 











198 



Journal of Agricultural Research 



Vol. XII, No. 4 



Tabids XII. — Influence of calcium nitrate on the fixation of nitrogen by Azotobacter in 

sterilized soil 





Treat- 
ment (ni- 
trate in 
100 gm. of 
dry soil). 


Total nitrogen in 100 gm. of dry soil. 


Nitrogen 


Culture No. 


Inoculated. 


Uninoculated. 


fixed in 
100 gm. of 




Found. 


Average. 


Found. 


Average. 


dry soil. 




Mgm. 













10 

10 

10 

10 

10 

10 

50 

50 

SO 

SO 

SO 

50 

200 

200 

200 

200 

200 

200 


Mgm.. 
133-0 
^33- (> 
133-3 
133-0 
134.2 

133-4 
137.0 
137.0 
136.5 
136.5 
137-0 

137-5 
148. 
148.5 
149.0 
148.5 
149-0 
148. 
173-0 
173.0 
174.0 

173-5 
173-0 
174.0 


Mgm. 
\ ^33- 3 

[ ^33- 5 
136. 8 

I 137- 
[ 148. 5 

148. 5 
1 173- 7 

173- 5 


Mgm. 
\ 131- 
1 131- 
[ 132- 


Mgm. 
\ 131- 3 


Mgm. 




2. 








] 








\ 2. 2 








J 




[ 135- 

1 134- 

135- 


[ 134- 7 






2. I 








1 








2.3 








J 


e 


[ 140. 5 

141. 

I 140. 5 


i 140. 7 




e 


7.8 


c 




6 


1 


6 






7-8 


6 






] ' 


7 


f 163. 

164. 

I 164- 5 


j 163. 8 




7 


9. 9 


7 




8 


1 


8 . .... 






1 .. 


8 















Table XIIL — Influence of calcium nitrate on the fixation of nitrogen by Azotobacter in 

unsterilized soil 





Treat- 


Total nitrogen in 100 gm. of dry soil. 


Nitrogen 


Culture No. 


ment (ni- 
trate in 
100 gm. of 
dry soil). 


Inoculated. 


Uninoculated. 


fixed in 
100 gm. of 




Found. 


Average. 


Found. 


Average. 


dry soil. 


I 


Mgm.. 













10 

10 

10 

10 

10 

10 

SO 

50 

50 

so 

50 

50 

200 

200 

200 

200 

200 

200 


Mgm. 
134-5 
136. 

136.5 
135.0 
135.5 
135-5 
138-5 
138.0 
139.0 
138.0 
137-5 
138.5 
151- 5 
152.0 
151. 
150.0 

151-5 
151.0 
177.0 
178.0 
176. 

176.5 
177.0 
178.0 


Mgm. 

135- 7 

[ 135- 3 

138. 5 

138. 

151-5 
i 150. 8 
[ 177-0 
[ 177- 2 


Mgm. 
1 134. 
1 ^33- 5 
[ 132. 
f 


Mgm,. 

\ ^33- 2 


Mgm. 


I 


2- 5 


1 




2 




2 







I ^' '■ 


2 


[ 






■^ 


[ 133- 5 

133- 

[ ^33- 


[ ^33- 2 




3 


5. 3 


2 




4 


1 


4 


1 




\ 4-8 


A 






J 


5 


[ 140. 5 
141. 

I 141. 5 
f 


\ 141.0 




c 


10.5 


c 


6 




6 






9-8 


6 






7 




f 164. 
165. 
[ 164. 
f 


\ 164- 3 




7 


12. 7 


7 


8 


1 


8 






1 12.9 


8 


|.. ....... 













Jan. 28. 1918 Nitrogen-Assimilatifig Boctena 199 

It will be seen at a glance that a greater relative increase in nitrogen 
fixation in the presence of nitrates occurred in the soil cultures than on 
the agar films. But in the latter instance the amount of nitrogen as- 
similated in the absence of mistakes is far in excess of that assimilated 
in the soil cultures under similar conditions. The amount of nitrogen 
fixed in the soil cultures is surprisingly low, but as relative increases or 
decreases are desired this does not materially influence the results. 

The influence of sodium nitrate on the fixation of nitrogen by pure 
cultures of Azotobacter in sterilized and unsterilized soil is brought out 
very clearly in the figures of Tables X and XI. In both cases, where 
no nitrate was added, an equal fixation of nitrogen occurred. Where 10 
mgm. of nitrate were added to 100 gm. of soil, slightly more nitrogen was 
assimilated in the unsterilized soil than in st'Crilized. The reverse seemed 
to be true when 50 mgm. of nitrate were added. But in the presence of 
150 mgm. of nitrate, the fixation by the pure culture in sterilized soil 
did not increase materially in comparison with that which occurred in 
the 50 mgm. of nitrate concentration. Evidently the maximum fixation 
under these conditions had been reached. The gain in the unsterilized 
soil at the highest concentration of nitrate studied almost doubled the 
amount fixed in the pure culture. It appears evident that the presence 
of sodium nitrate causes a greater fixation of nitrogen in unsterilized soil 
than it does under similar conditions in sterilized soil inoculated with 
Azotobacter. 

In the case of calcium nitrate, somewhat comparable results were ob- 
tained. The fixation where no nitrate was added was equivalent to 
that obtained in the controls for the sodium nitrate. Where nitrate 
was added in amounts equal to 10 mgm. of nitrate in 100 gm. of soil, an 
increased fixation w^as obtained in the unsterilized soil, but practically 
no increase occured in the pure culture in sterilized soil. Fifty mgm. 
of nitrate in 100 gm. of soil produced an increase in fixation. In the 
highest concentration of calcium nitrate the difference in nitrogen fixed 
between the pure culture in sterilized soil and unsteriUzed soil was not so 
great as in the case where sodium nitrate was used. 

In the sterilized soil where the two nitrates were present in equal 
amounts it can be seen that more fixation took place in the presence of 
sodium nitrate. The difference is not marked, but it exists neverthe- 
less. It will be remembered that calcium nitrate had a detrimental 
effect on nitrogen fixation by Azotobacter on agar films. However, in 
soil cultures this same nitrate stimulated Azotobacter to an increased 
assimilation of nitrogen. This difference is not suprising as it has been 
shown repeatedly that bacterial activities in soil and in artificial cultures 
are not always comparable. 

From the results of the experiments performed with reference to the 
influence of nitrates in soil on the fixation of nitrogen therein, it appears 



200 Journal of Agricultural Research voi. xii. no. 4 

evident that in pure cultures both sodium and calcium nitrates in the 
amounts studied produced an increase in the amount of nitrogen fixed. 
The sodium salt stimulated this process to a slightly greater extent than 
did the calcium salt. In unsterilized soil nitrates exerted the same 
action but to a more marked extent. The amount of nitrogen fixed 
under these conditions was generally in excess of that fixed under similar 
conditions in sterilized soil inoculated with a pure culture of Azotobacter. 

Such large relative increases in total nitrogen in the soil in the presence 
of nitrates would not normally take place under field conditions for here 
no accumulations of nitrate occur in quantities sufficiently large enough to 
influence this process. 

Summing up all the experiments performed in relation to the influence 
of nitrates on the fixation of atmospheric nitrogen by Azotobacter, it 
appears that the increase in total nitrogen in the presence of these salts 
is by no means comparable to the increase in the number of organisms 
in sterilized soil under the same conditions. An increase in the number of 
Azotobacter does not mean a parallel increase in the amount of nitrogen 
fixed. 

INFLUENCE OF AZOTOBACTER ON NITRATES IN SOLUTION 

Attention has been thus far directed toward the influence exerted 
by nitrates on the growth and nitrogen-assimilating power of Azotobacter. 
The following points are now to be considered: Do the nitrogen-fixing 
bacteria reduce nitrates to nitrites and ammonia? Is there an increase 
or decrease in the amount of organic nitrogen as a result of the presence 
of nitrate in the medium? 

Beijerinck and Van Delden (5) found that Azotobacter chroococcum 
reduced nitrate directly to ammonia. Stoklasa (44, p. 492-503) 
studied the changes in a nutrient solution containing 0.2 per cent 
of sodium nitrate inoculated with Azotobacter. He found under an- 
aerobic conditions that the nitrate was largely reduced to nitrite and 
ammonia and that a very small amount of organic nitrogen was formed. 
Under aerobic conditions there was more nitrite formed than under 
anaerobic conditions and very little ammonia or oganic nitrogen. He 
concluded, therefore, that Azotobacter did not fix atmospheric nitrogen 
in the presence of nitrates. 

The following experiments were performed in an endeavor to answer 
the questions raised in the initial paragraph of this section. To Erlen- 
meyer flasks of 500-c. c. capacity, containing loo-c. c. portions of mannit 
solution, sodium and ammonium nitrates were added in amounts equiva- 
lent to 150 mgm. of nitrate in loo c. c. of the solution. Nine flasks were 
prepared for each nitrate and the same number for the controls containing 
no nitrate. The flasks and contents were sterilized at 10 pounds pres- 
sure for 30 minutes. After cooling, six of each set were inoculated, three 



Jan. 28, 1918 



Nitrogen- A ssimilating Bacteria 



201 



with strain A and three with strain B, and the remaining three were left 
uninoculated to serve as controls. The flasks were incubated at 28° C, 
for 21 days. The total weight was maintained throughout the experi- 
ment by the addition of sterile distilled water from time to time. At the 
end of three weeks the contents of each set of triplicate flasks were poured 
together and 50-c. c. samples drawn for analysis. Nitrate ammonia and 
total nitrogen were determined as given under "Methods." The results 
are shown in Tables XIV, XV, and XVI. 

Table XIV. — Influence of Azotobacter on nitrates in solution, giving the quantity of 

nitrate lost 





Treatment (ni- 
trate in 100 
c. c. of me- 
dium). 


Nitrate in loo c. c. of medium. 


Cul- 


Strain A. 


strain B. 


ture 
No. 


Inoculated. 


Uninoculated. 


Nitrate 
lost. 


Inoculated. 


Uninoculated. 






Found. 


Aver- 
age. 


Found. 


Aver- 
age. 


Found. 


Aver- 
age. 


Found. 


Aver- 
age. 


lost. 






Mgm. 

0.00 

.00 

80.9 
80.6 

100.3 
102. 1 


Mgm. 
1 0.00 

jso. 75 

>I0I.2 


Mgm. 
( 0.00 
\ .00 

/iSO- 4 
li5i-3 

/i49- 6 
\i50. 


Mgm. 
> 0. 00 

}i50.8 

}i49-8 


Mgm. 
0.00 

0— 70.0s 

a— 48. 60 


Mgm,. 
f 0.00 
\ 00 

/105. 6 
1105. 2 

/l3l- I 
\i30. 7 


Mgm.. 
1 0.00 

}ios-4 
1 130.9 


Mgm. 
f o-oo 

\ .00 

/iSO- 4 
\iSi-3 

/149- 6 


Mgm. 
> 0. 00 

|iS0.8 

}i49.8 


Mgm. 






0.00 


lo-iS 


isogm.of NO3 
as sodium ni- 
trate 






. . do ..... 


6—45-40 


19-27 
19-27 


isomgm.of NO3 
as ammonium 
nitrate 

do 


C-18.90 











o Strong NO2 r,eaction. 



b Medium NO2 reaction. 



c Slight NO2 reaction. 



Table XV. — Influence of Azotobacter on nitrates in solution, giving the quantity of 

ammonia produced 





Treatment (ni- 
trate in 100 
c. G. of me- 
dium). 


Nitrogen as ammonia in 100 c. c. of medium. 


Cul- 


Strain A. 


Strain B. 


ture 
No. 


Inoculated. 


Uninoculated. 


Am- 
monia 

pro- 
duced. 


Inoculated. 


Uninoculated. 


Am- 




Found. 


Aver- 
age. 


Foimd. 


Aver- 
age. 


Found. 


Aver- 
age. 


Found. 


Aver- 
age. 


pro- 
duced. 


1-9 

1-9 

10-18 




Mgm. 

0.20 

. 10 

2.00 
1.80 

13-90 
13- 9S 


Mgm. 
I 0.15 

I I- 90 

}i3-97 


Mgm.. 
( 0.00 
\ .00 

f-.IO 

\ . 20 

/ 13-90 
I 13-90 


Mgm. 
>• 0. 00 

}i3-90 


Algm. 
0.15 

1.85 

.•07 


Mgm. 
( o^oo 
\ .20 

f 2.20 

I 2-40 

/ 13-80 
1 13-80 


Mgm. 
\ 0. 10 

} s-30 
}i3-8o 


Mgm. 
f 0.00 
\ .00 

f-.io 
\ .20 

/ 13-90 
I 13-90 


Mgm. 
} 0. 00 

}i3-90 


Mgm. 






isomgm.of NO3 
as sodium ni- 
trate 






do 


2- 25 


19-27 
19-27 


isomgm.of NO3 
as ammonium 
nitrate 

do 


. 10 



202 



Journal of Agricultural Research 



Vol. XII, No. 4 



TablB XVI. — Influence of Azotobacter on nitrates in solution, giving the quantity of 

nitrogen fixed 





Treatment (ni- 
trate in 100 
c. c. of me- 
dium). 


Total nitrogen in 100 c. c. of medium. 


Cul- 


Strain A. 


Strain B. 


ture 
No. 


Inoculated. 


Uninoculated. 


Nitrogen 
fixed. 


Inoculated. 


Uninoculated - 


Nitro- 




Found. 


Aver- 
age. 


Found. 


Aver- 
age. 


Found. 


Aver- 
age. 


Foimd. 


Aver- 
age. 


gen 
fixed. 


1-9 

1-9 

lO-lS 




Mgm. 
5.00 
5.00 

22. 50 
22.60 

47.00 
46.90 


Mgm- 
I S-oo 

I22.5S 

}46. 95 


Mgm. 
f 2.60 
I 2- 70 

/ 14.00 
\ 14.20 

/ 43' 20 
I 42.90 


Mgm. 
}2.6s 

}i4- 10 

|43-os 


Mgm. 
2-35 

8-45 

3- 90 


Mgm. 
f S-oo 
I 5- 10 

/ 28.00 
\ 27- So 

f 48. TO 
\ 48. 20 


Mgm.. 
I S-os 

J27.90 

}48. IS 


Mgm. 
f 2.60 
I 2.70 

f 14.00 
\ 14.20 

/ 43-20 
I 42.90 


Mgm.. 
} 2-6s 

|i4. 10 

}43-0S 


Mgm. 




2.40 


iSomgm.ofNOs 
as sodium ni- 
trate 

do 


13- 80 


19-27 
19-27 


isomgm.of NO3 
as ammonium 

nitrate 

do 


S-io 







Table XIV showing the effect on the^ total nitrate content will be 
discussed first. Strain A differed widely from strain B in its ability to 
reduce nitrates. It will be noted that strain A reduces nitrate more 
readily than strain B in the presence of both sodium and ammonium 
nitrate. In order to determine the nature of the reduction of the nitrates, 
qualitative and quantitative tests were made. The reduction of nitrates 
by Azotobacter takes place with the formation of nitrites as shown in 
Table XIV. Strain A effected a strong reduction of nitrate to nitrite 
with both sodium and ammonium nitrate. Strain B also reduced nitrate 
to nitrite, but to a lesser degree than did strain A. 

An inspection of the data in Table XV indicates that the reduction of 
nitrates ceased with the formation of nitrite, since no appreciable amounts 
of ammonia were produced by either strain of Azotobacter. 

In regard to the fixation of atmospheric nitrogen by these strains of 
Azotobacter, it was found that nitrogen was assimilated both in the 
presence and absence of nitrate. In the presence of nitrate there was a 
large increase in the total organic nitrogen. Sodium nitrate stimulated 
both strains, although strain B fixed the larger amount. Similar results 
were obtained when the fixation of nitrogen on agar films was studied. 
In the presence of ammonium nitrate the amount of nitrogen fixed was 
considerably decreased, but the amount fixed was in excess of the control 
cultures containing no nitrate. It seems evident that sodium and 
ammonium nitrate in the amounts studied did not prevent the fixation 
of atmospheric nitrogen. In fact, the presence of these salts seemed to 
stimulate the process. 

Under aerobic conditions both strains of Azotobacter studied caused a 
reduction in the total amount of nitrate present in the solution. This 
reduction may be accounted for in two ways: (i) The reduction of 
nitrate to nitrite and (2) the assimilation of nitrate by the organisms. 
Practically no ammonia was formed under the conditions of these experi- 
ments. These results agree with those of Stoklasa. However, in con- 



Jan. 28, i9i8 Nitrogen-Assimiloting Bacteria 203 

trast to the work of Stoklasa, both strains of Azotobacter assimilated 
more atmospheric nitrogen in the presence of nitrates in solution than 
in the absence of these salts. 

INIfLUENCE OP NITRATES ON THE PRODUCTION OP PIGMENT BY AZOTOBACTER 

It has already been noted in the experiments dealing with the effect 
of nitrates on the fixation of atmospheric nitrogen on agar films that 
nitrates favor pigment production. This was true in the case of both 
strains of the Azotobacter. 

Moreover, it has been observed by other investigators that Azotobacter 
when grown in the presence of nitrate will produce a darker pigment 
than when grown in its absence. Beijerinck (4, p. 575) states that 
Azotobacter in pure culture will form a dark-bro\vn pigment in the 
presence of glucose and a small amount of nitrate. Sackett {43) found 
that nitrate caused an increase in pigment production by Azotobacter. 
In media without the nitrate the pigment formation was materially 
decreased and in some cases practically eliminated. He also noted that 
the amount of nitrate present has a direct influence on the intensity of 
the pigment formation. He found that when sodium nitrate was added 
to a suitable medium to give a content of 0.0, o.oi, 0.03, 0.05,0.08, o.i, 
0.3, and 0.5 per cent, with glucose used as the source of energy, the 
organisms produced pigment. Streak inoculations were made, and 
after 14 days' incubation he found that the maximum of color was 
obtained at 0.05 to 0.08 per cent and that greater concentrations did not 
increase the intensity of the brown-black pigment. From his results it 
is evident that sodium nitrate caused an increase in pigment formation 
by azotobacter. 

In order to determine the possible effect of potassium, sodium, and cal- 
cium nitrate on pigment formation with strains A and B, the following 
experiment was performed. 

Under normal conditions on mannit agar free from nitrate strain A 
produced little or no pigment even after three weeks' growth. At the 
end of this time dirty-yellow streaks occurred throughout the growth, 
but no brown pigment was produced. However, with strain B at the 
end of two or three weeks a decided brown to brown-black pigment was 
produced in the absence of nitrate. 

Agar slope cultures containing increasing amounts of potassium, so- 
dium, and calcium nitrate, as indicated in Table XVII, were prepared. 
These were inoculated with both strains of Azotobacter and incubated 
at 28° C. for 10 days. Daily observ^ations were made for first evidences 
of pigment formation. In some of the cultures of strain A growing on 
media containing calcium nitrate this pigmentation was observed as 
early as 48 hours subsequent to inoculation. The following day pig- 
mentation developed in strain B. The cultures on the potassium and 
sodium-nitrate media began to show evidence of pigmentation in four to 
six days. The final results, obtained after 10 days' incubation, are found 
in Table XVII. 



204 



Journal of Agricultural Research 



Vol. XII, No. 4 






<; 

^ 






8 



a o 



55^ 



;-« 



SPS Qpq 









« 



a „ 

boooooo." 
tS 



o 
•=i S' 



o 

OtQ o 






O lU 



_ ^ o o 

13 -i-"d .S 'O "d 

t3 



^ p 

bo'- 



.^^ 






_ ^ _ o o 

-*-" "d .^i Td "d "O 



.OOioOOOOO 

a Hcsvoomoo 

C H H N ro 



H N fO 'J' >oo r— 00 



Jan. 28. i9i8 Nitrogefi-Assiniilating BocteHa 205 

A general idea may be gained from Table XVII concerning the relative 
increase in pigment formation in the presence of the nitrates. A study 
of the table gives a fair idea of the relative differences in pigment pro- 
duction. 

Very interesting results were obtained with strain A. It will be seen 
from Table XVII that no pigment was produced in the control culture 
after 10 days, while in the presence of nitrates pigmentation was noted. 
The intensity of the pigment varied with the increase of nitrate up to 150 
mgm. Beyond 150 mgm. there was no increase. Potassium and sodium 
nitrate did not exert such a decided influence on pigment production as 
calcium nitrate. The latter salt produced an intense dark-brown to 
brownish-black pigment. 

In the case of strain B the influence of nitrate was not so pronounced 
since this strain normally produced considerable pigment in the absence 
of nitrates. Potassium and sodium nitrate caused a slight increase in 
pigment formation. Here, again, the calcium salt brought about most 
pronounced increase. However, the relative increase in pigment forma- 
tion in strain B was not so pronounced as in strain A. 

Where the nitrate was present, a much more spreading growth was 
obtained. A heavy bacterial growth accumulated at the base of the slope 
except in the two cultures in which the highest concentrations were used. 
In the latter instances the accumulation was less than those in cultures 
growing on media containing no nitrate. Although the original inocula- 
tion could not be made absolutely uniform, so far as number of organ- 
isms was concerned; nevertheless it was evident that on those slopes 
containing 10, 25, 50, and 100 mgm. of nitrate in 100 c. c. of the medium 
a much more abundant growth was obtained than on those slopes free 
from nitrate. Here, again, it is seen, in a rough, comparative way, that 
the smaller amounts of nitrates caused an increase in the number of 
Azotobacter. 

The results of this work on pigment production are quite in accord with 
those of Sackett. Potassium, sodium, and especially calcium, nitrates 
in varying amounts increase pigment formation by Azotobacter with an 
increase in nitrate concentration. This effect is especially marked in 
strain A, which under normal conditions does not produce any pigment. 

INFLUENCE OK NITRATES ON THE FORMATION OF VOLUTIN BODIES IN AZOTOBACTER 

The presence of volutin bodies, or metachromatic granules, in Azoto- 
bacter has been shown by Bonazzi (7). These substances, according to 
Meyer (54, p. 238), are reserve food materials other than fat droplets, 
glycogen, and similar substances reacting with iodin stain which occur 
in the cytoplasm of the cells of various bacteria. With Millon's reagent 
they give no reaction. He believes that these bodies are composed of 
nucleic-acid compounds, but are not nuclear proteids. 



2o6 



Journal of Agricultural Research 



Vol. XII, No. 4 



In connection with the foregoing investigations concerning the influ- 
ence of nitrates on pigment formation by Azotobacter, it was thought 
that some results of cytological interest might be obtained in regard to 
the effect of varying amounts of nitrates on the volutin bodies. 

Slope cultures of mannit agar were prepared containing the different 
nitrates as indicated in Table XVIII. These slopes were inoculated 
with both strains of Azotobacter and incubated at 28° C. for 10 days. 
At the end of this time each culture was stained and examined micro- 
scopically. The following method was used for demonstrating the 
presence of the volutin bodies. The organisms to be examined were 
air dried on a glass slide and then fixed in the flame of a Bunsen burner. 
The preparation was then flooded with a i to 10 aqueous solution of 
methylene blue (Merck's) prepared by adding 10 c. c. of a saturated 
aqueous solution of methylene blue to 90 c. c. of distilled water. The 
stain was washed off after five minutes with a i per cent solution of 
sulphuric acid and immediately rinsed in distilled water. The prepa- 
ration was dried and examined with the oil-immersion objective. The 
volutin bodies appeared within the cytoplasm as very dark blue dots, 
the outline of the cell wall was a lighter blue, while the cell net^work 
was stained a very light blue. 

Guignard's stain ^ was also used to demonstrate the presence of the 
volutin bodies. Fresh smears on a glass slide were fixed over 10 per 
cent osmic acid for three minutes. The preparation was then air-dried 
and fixed to the slide by rapidly passing the latter a few times through 
a Bunsen burner. The preparation was covered with the stain which 
was allowed to react for five minutes. The stain was then washed off 
with distilled water, dried, and examined with the oil-immersion objec- 
tive. The outline of the cell as well as the net work within-was stained 
light purple. The granules within the cytoplasm were a reddish purple. 
The results are given in Table XVIII. 



Tabi^e XVIII.- 



-Influence of nitrates on the formation of volutin bodies in Azotobacter 
in 10 days 





Treat- 
ment 
(nitrate 
in 100 
c. c. of 

me- 
dium). 


Strain A. 


Strain B. 


Culture 
No. 


Potassium 
nitrate. 


Sodium 
nitrate. 


Calcium 
nitrate. 


Potassium 
nitrate. 


Sodium 
nitrate. 


Calcium 
nitrate. 




Mgm. 

10 

25 

so 
100 
ISO 
200 
300 


Present.o. . . . 

do.o 

do.o 

do.o 

do.o 

do.o 


Doubtful.... 

Present 

do.o 

do.ft 

do.ft 

... do ft 


Doubtful. . . . 


Present 

do 


Doubtful.... 

Present o 

do.ft 

do.o 

do.ft 


Present. 
Do. 


2 




do.o 

Doubtful. . . . 


do.o 

do." 

do ft ... 






Do.o 
Do 


s 


6 


do 


do ft 


do ft 


Do ft 


7 


do. 6 


do.ft 


. . do ft 


.. do.ft 


.... do.ft ... 


Do 


8 


Ao.b 


.... do.6 . 


do 


doi>. ... 


.. do ft 


Do ft 

















o Representing an approximate average of two volutin bodies per cell. 

b Representing an approximate average of four volutin bodies per cell. 

' Guignard'sstain. Fifty c. c. of 2 per cent fuchsin in i per cent acetic acid; 40 c. c. of 0.2 per cent methyl 
green in i per cent acetic acid; i c. c. of glacial-acetic acid. Distilled water was used in making the i per 
cent acetic-acid solution. 



Jan. 28, 1918 



NitYogen-A ssimilating Bacteria 



207 



It will be seen that all three nitrates exerted considerable influence 
on the formation of volutin bodies. Not only was the number of bodies 
increased, but also the size. The relative increase in size of the granules 
was much more marked than was the numerical increase. In Azoto- 
bacter cells grown on mannit agar containing no nitrate the number of 
volutin bodies in each cell averaged about two ; in the presence of nitrate 
four to five volutin granules were found. The greatest increase in num- 
ber, as well as size, occurred where the nitrate concentration was highest. 
With both strains sodium nitrate apparently caused the greatest increase. 
This was true in the lower as well as in the higher concentrations. The 
volutin bodies in strain B seemed to respond to the presence of nitrates 
more noticeably than did those of strain A, especially in the presence of 
potassium nitrate. It is evident that nitrates of potassium, sodium, 
and calcium cause an increase in the number and size of volutin bodies 
in Azotobacter cells. 

Do these salts tend to hasten the appearance of these bodies, or do 
they at first retard their development? The following experiment was 
carried out in an endeavor to determine this point. Only sodium nitrate 
was used, since this particular salt proved most beneficial to the forma- 
tion of volutin bodies. Agar slopes were prepared containing the different 
amounts of nitrate as indicated in Table XIX. The cultures were incu- 
bated at 28° C. and examined daily for the presence of volutin bodies. 
The methylene blue — i per cent sulphuric acid — method of staining was 
employed. The iresults of the experiment are given in Table XIX. 

Table XIX. — Influence of sodium nitrate on the rate of formation of volutin bodies. in 

Azotobacter 





Time. 




Nitrate in 100 c. 


c. of medium. 






Strain A. 


Strain B. 




oMgm. 


25 Mgm: 


100 Mgm. 


300 Mgm. 


Mgm. 


25 Mgm. 


100 Mgm. 


300 
Mgm. 




Day. 


Absent... 

Present". 

...do.a 

...do." 


Absent.. . 

Present «. 
...do.a.... 
. . .do.o. . . . 


Doubtful. 

Present a. 
...do.".... 
...do. 6... 


Doubtful. 

...do.6... 
...do.6... 
...do.b... 


Absent... 
...do 

Present". 
. . .do.o 


Absent... 

Present". 
...do.b... 
...do.b... 


Doubtful. 

...do.ft... 
...do.b... 
...do.b... 


Dbtful. 




Do.b. 


3-- 
4.. 




Dob. 
Do.b. 



o Representing an approximate average of two volutin bodies per cell. 
b Representing an approximate average of four volutin bodies per cell. 

A study of Table XIX shows that it is rather doubtful whether the 
nitrate present tended to hasten the appearance of the volutin bodies. 
No convincing evidence has been presented for or against this statement. 
No granules were seen^in the first day's growth of strain A, although 
the next day they were present in all four cultures. In strain B more 
convincing proof is furnished that the sodium nitrate hastened the 
appearance of these reserve food substances. The volutin bodies were 
not present in the control and lowest nitrate concentration cultures 
the first day, but they were very noticeable in the culture containing 
the highest concentration of nitrate and doubtful in the remaining one. 
On the second day volutin bodies were present in all cultures grown on 



208 



Journal of Agricultural Research 



Vol. XII, No. 4 



nitrate media, v/hile the control culture was still free from them. The 
third day showed the presence of volutin bodies in all four cultures. 
Strain B offers the better proof that sodium nitrate tends to hasten the 
appearance of volutin bodies in the cells of Azotobacter. Further 
experiments were not made in an endeavor to determine what influence 
nitrates might have on the cytology of the Azotobacter cell. The brief 
studies reported here were made in connection with the pigment forma- 
tion experiments, but do not bear any particular relation to them. The 
increase in number and size of volutin bodies may bear some relation to 
the increased amount of nitrogen fixed or assimilated by Azotobacter in 
the presence of nitrates. 

INFLUENCE OF NITRATES ON BACILLUS RADICICOLA 

INFLUENCE OP NITRATES ON THE GROWTH AND REPRODUCTION OP BACILLUS RADICICOLA 

IN STERILIZED SOIL 

One hundred and fifty gm. (dry weight) of the soil were weighed 
into 500-c. c. Hrlenmeyer flasks and the nitrates added as indicated 
in Tables XX-XXII. Duplicate cultures for each amount of nitrate 
were prepared. One per cent of mannit (in 5 c. c. of distilled water) 
was also added. The flasks were kept at room temperature for one 
day and the contents then thoroughly mixed. The flasks were steri- 
lized at 1 5 pounds' pressure for three hours. Upon cooling they were 
inoculated with 5 c. c. of a suspension of Bacillus radicicola in sterile 
distilled water. The number of bacteria in the inoculum was deter- 
mined. The moisture content was then approximately 18 to 20 per 
cent. The flasks were incubated at 28° to 30° C. and mannit-agar 
plates poured at the end of one and two weeks. The results of these 
experiments are given in Tables XX, XXI, and XXII, in which each 
figure represents an average of duplicate plates. 

Table XX. — Influence of potassium nitrate on Bacillus radicicola in sterilized soil 



Culture No. 


Treat- 
inent (ni- 
trate in 
100 gm. of 
dry soil). 


Number of organisms in i gm. of dry soil. 


At begin- 
ning. 


After I week. 


Relative. 


After 2 weeks. 


Relative. 


I 

2 


Mgm. 





10 

10 

25 

25 

50 

50 

100 

100 

150 

150 

200 

200 

300 

300 


10, 670 
10, 670 
10, 670 
10, 670 
10, 670 
10, 670 
10, 670 
10, 670 
10, 670 
10, 670 
10, 670 
10, 670 
10, 670 
10, 670 
10, 670 
10, 670 


680, OOQ 
825, 000 

2, 195, 000 

3, 410, 000 
3, 900, 000 
3, 885, 000 
1,555,000 
I, 585, 000 

445, 000 
320, 000 
375, 000 
330, 000 

135, OOO 

170,000 

45, 000 
50, 000 


Per cent. 
100 

I 372 

} 517 

1 208 

} ^^ 
} 46 

> 2Q 
} 6.3 


f 8, 015, 000 
\ 7, 000, 000 
f 14, 600, 000 
\ 11,050,000 

r IK. AOO. 000 


Per cent. 
> 100 

1 


■2 


4 


C 


6 


\ 14, 800, 000 1 1 


7 


f II, 500, 000 
\ 14, 400, 000 

f 2, 680, 000 
\' 3, 290, 000 

r 560, 000 
\ 790, 000 
f 90, 000 

I («) 
J 25, 000 

I 3°, 000 


} 173 
1 40 
} 8.9 

} - 
} -. 


8 


Q 


10 


II 


12 


JT. 


14 


IC 


16 





a Contamination. 



Jan. 98, 191S 



Nitrogen- A ssimilating Bacteria 



209 



Table XXI. — Influence of sodium nitrate on Bacillus radicicola in sterilized soil 





Treat- 
ment (ni- 
trate in 
100 gm. of 
dry soil). 


Number of organisms in i gm. of dry soil. 


Culture No. 


At begin- 
ning. 


After I week. 


Relative. 


After 2 weeks. 


Relative. 




Mgm. 





10 

10 

25 

25 

50 

50 

100 

100 

150 

150 

200 

200 

300 

300 


i5> 500 
15, 500 
i5> 500 
i5,.5oo 
15. 500 
15. 500 
15. 500 
15, 500 
15, 500 
i5> 500 
15, 500 
15. 500 
i5> 500 
15. 500 
i5> 500 
15. 500 


I, 500, 000 

1, 250, 000 

2, 560, 000 
3, 000, 000 
6, 150, 000 
5. 375. 000 

4, 850, 000 

5, 570, 000 
2, 000, 000 
I, 850, 000 
I, 060, 000 

835, 000 
760, 000 
725, 000 
250, 000 
365, 000 


Per cent. 

> 100 

> 201 
} 418 
} 378 
\ 140 
} 69 

} ^"^ 
22 


/ 6, 750, 000 
\ 5, 950, 000 
f 10, 000, 000 
\ 12, 500, 000 
f 14, 650, 000 
\ 15, 700, 000 

\ 8, 500, 000 
f I, 520, 000 
\ I, 650, 000 
/ 850, 000 
\ 940, 000 
f 500, 000 
\ 620, 000 
f 150, 000 
\ 210,000 


Per cent. 


2 


> 100 








1 177 


e 




6 


> 240 


7 




8 


I 134 









} ^^ 








1 ^'^ 




1 8.8 


IJ. 


TC 


1 2.8 


16 







a Contamination. 
Table XXII. — Influence of calcium nitrate on Bacillus radicicola in sterilized soil 





Treat- 
ment (ni- 
trate in 
100 gm.of 
dry soil). 


Number of organisms in i gm. of dry soil. 


Culture No. 


At begin- 
ning. 


After I week. 


Relative. 


After 2 weeks. Relative. 




Mgm. 





10 

10 

25 

25 

50 

50 

100 

100 

150 

150 

200 

200 

300 

300 


oppppppppppppppP 

OOOOOOOOOOOOOOOO 


960, 000 

850, 000 

3, 650, 000 

3, 940, 000 

5, 500, 000 

6, 700, 000 
4, 000, 000 
3, 500, 000 


Per cent. 
100 

1 419 
} 674 
} 414 


1 
Percent. 

j 4,675,000 :\ ^^ 


2 


i 4,590,000 J 


1 


/ 6,000,000 1 


A 


\ 5,450,000 J 


C 


/ 10,650,000 1 


6 


I 14,700,000 J '^ 


7 


f 9,350,000 j1 

1 8,670,000 1/ ^95 


8 




1, 200, 000 1 ^g^ 

2, 050, 000 iJ 
S65.000JI ^^^ 

I, ot^o, 000 J 
375.000:1 
260, 000 ij "'-' 

35.000 1 

47,000 ,1 ^ ^ 


f 1,500,000 1 


10 


\ 1,750,000 j ^^ 


II 


f 765,000,1 


12 


\ 800,000 1 ^' 


1-2 


f 350,000 1 _ 
\ 300,000 / '■ 


14 


TC 


( 25,0001 

\ 40,000 J ' 


16 








1 



An inspection of all three tables reveals two marked differences from 
the results obtained in similar work with Azotobacter. First, it will be 
noted that nitrates do not appear to exert such a marked stimulating 
effect with B. radicicola as wath Azotobacter. The numerical increase 
due to the presence of the nitrate is clearly shown in the percentage 
columns. Second, it will be noted that B. radicicola does not seem to be 
so sensitive to higher concentrations of nitrates as does Azotobacter. 
In all instances at concentrations equivalent to 300 mgm. of nitrate in 



2IO 



Journal of Agricultural Research 



Vol. XII, No. 4 



loo gm. of soil the legume organisms were still alive, although present 
in numbers far below those of the control cultures. In all Azotobacter 
cultures no organisms survived this concentration. 

No one nitrate produced an excessive stimulation in comparison with 
the others. The calcium salt present as 150 mgm. of nitrate in 100 gm. 
of soil at the end of the first week gave the greatest stimulation for con- 
centrations of that amount. However, at the end of the second week 
this concentration had caused a marked decrease in the number of or- 
ganisms. In the case of all three nitrates the concentration representing 
25 mgm. of nitrate in 100 gm. of soil produced the greatest stimulation. 
This resulting stimulation also held true throughout the second week. 
The decrease in number below those of the control cultures, due to in- 
creasing concentrations of nitrate, began first in the presence of potassium 
nitrate at 100 mgm. of nitrate per 100 gm. of soil, then with sodium 
nitrate at 150 mgm., and lastly with calcium nitrate at 200 mgm. But 
the number of organisms present in the soil cultures containing sodium 
nitrate in amounts equivalent to 100 mgm. and calcium nitrate at 100 
mgm. at the end of the second week was below those of the control 
cultures. 

It therefore appears from these results that small amounts of potassium, 
sodium, and calcium nitrate stimulate the reproductive activity of B. 
radicicola. Concentrations of nitrates greater than those amounts which 
produced maximum stimulation cause a decrease in the number of or- 
ganisms. The highest concentration of nitrate studied did not entirely 
prevent the growth of the bacteria, but it reduced the number of organ- 
isms far below those contained in control cultures where no nitrates 
were added. 

Ammonium nitrate was also employed. The soil cultures were pre- 
pared as already described and inoculated with B. radicicola. The cul- 
tures were incubated at 28° to 30° C. and counts were made at the end 
of one and two weeks' time. The results of the study with ammonium 
nitrate are dven in Table XXIII. 



Table XXIII. — Influence of ammonium nitrate on Bacillus radicicola in sterilized soil 





Treat- 
ment (ni- 
trate in 
100 gm.of 
dry soil). 


Number of organisms in i gm. of dry soil. 


Culture No. 


At begin- 
ning. 


After I week. 


Relative. 


After 2 weeks. 


Relative. 


I 


Mgm. 





25 

25 

100 

100 

200 

200 


P P P P P P P P 
OOOOOOOO 


850, 000 
765, 000 

2, 500, 000 

3, 050, 000 

I, 350, 000 

I, 050, 000 

700, 000 

655, 000 


Per cent. 
\ 100 

} 343 
} 148 
} 84 


/ , I, 365, 000 
\ I, 400, 000 

r 5, 060, 000 
\ 4, 320, 000 
/ 1, 030, 000 
\ 950, 000 
1 635, 000 

\ 605, coo 


Per cent. 
\ 


2 


> 100 


7, 


1 


4 


\ 338 


C 


1 


6 


/ ^' 


7 


1 


8 


} ' 45 







Jan. 28, i9i8 Nitrogen-Assimiloting BacteHa 211 

From the results as a whole it appears that it is the nitrate radical 
and not the combined salt which causes the increase in the number of 
B. radicicola when small amounts of nitrates are present. A stimulation 
occurred, resulting in an increase in number which is quite comparable 
to that obtained with potassium, sodium, and calcium nitrates. The 
highest concentration of ammonium nitrate used did not appear to have 
such an inhibiting effect as did the corresponding concentrations of the 
three other salts. 

Throughout the work with B. radicicola in sterilized soil compara- 
tively low numbers of these organisms were found. Whether or not 
this depression was due to toxic substances formed as a result of steriliza- 
tion can not be stated. If this decrease in numbers as a result of the 
presence of toxic substances is true, it is very evident that the detrimental 
effect had not become materially lessened at the end of the incubation 
period. However, in any event the validity of the outcome is not im- 
paired, since comparative and not absolute data are of importance and 
since in all probability the same conditions obtained throughout the 
cultures. 

It seems certain from the results of these studies on the effect of 
potassium, sodium, calcium, and ammonium nitrates on the growth of B. 
radicicola in sterilized soil that small amounts of nitrate stimulate the 
growth of the organisms. It is also shown that B. radicicola is much 
more resistant than Azotobacter to higher concentrations of potassium, 
sodium, calcium, and ammonium nitrates. 

INFLUENCE OP BACILLUS RADICICOLA ON NITRATES IN SOLUTION 

The series of soil culture experiments just discussed served to give an 
idea concerning the effect of nitrates on the legume organism. It was 
found that in small amounts nitrates stimulated the bacteria to increased 
reproduction. But no study was made as to the effect of Bacillus radicicola 
on the nitrate. Does the organism break up the nitrate, reducing it to 
nitrite or ammonia ? Does it cause any loss in nitrate when grown in a 
solution containing that salt? Beijerinck (2, p. 762) as a result of 
physiological experiments with B. radicicola, states that the organism does 
not reduce nitrate. Prucha (41) also states that B. radicicola does not 
reduce nitrates. However, Zipfel (49) found that B. radicicola will reduce 
nitrates to nitrites but not to ammonia. 

The following experiments, somewhat similar to those already cited in 
relation to Azotobacter, were carried out in an endeavor to answer these 
questions. 

To twenty 500-c. c. Erlenmeyer flasks containing 200 c. c. of mannit 
solution, potassium, sodium, calcium, and ammonium nitrates were 
added as indicated in Tables XXIV, XXV, and XXVI. Quadruplicate 
flasks were prepared for each concentration of nitrate and for the control 
cultures without nitrate. The flasks and contents were sterilized at 



212 



Journal of Agricultural Research 



Vol. Xn, No. 4 



lo pounds' pressure for 30 minutes. After cooling, two of each set of four 
flasks were inoculated with 5 c. c. of a suspension of B. radicicola in 
sterile distilled water. The remaining two flasks of each set (uninocu- 
lated) served as controls. The flasks were incubated at 28° C. for 2 1 days. 
The total weight of the flasks was maintained throughout the incuba- 
tion period by the addition of sterile distilled water from time to time. 
At the expiration of the period of incubation the nitrate, ammonia, and 
total nitrogen contents were determined as given under "Methods used 
in experiments." The contents of the duplicate inoculated flasks were 
poured together and 50 c. c. samples drawn for analysis. The same 
procedure was followed in the case of the uninoculated flasks. The 
results are given in Tables XXIV, XXV, and XXVI. 

Table XXIV. — Influence of Bacillus radicicola on nitrates in solution giving the quantity 

of nitrate lost 





Treatment (nitrate in loo c. c. of medium). 


Nitrate in loo c. c. of medium. 




Cul- 
ture 
No. 


Uninoculated. 


Inoculated. 


Nitrate 
lost. 




Found. 


Average. 


Foimd. 


Average. 




I 


None 


Mgm. 
0. 00 
. 00 

151- 4 
151. 
148.8 
148.8 
154-8 
155-6 
151- 4 
151. 6 


Mgm. 
> 0. 00 

}i5i- 2 

[148. 8 
}l55-2 

}i5i-5 


Mgm. 
( 0. 00 
\ . 00 

/117-O 
I117.O 
flI4. 4 
\ii4. 
/ 76.6 
I 76.8 
fi42. 6 
\i42. 6 


Mgm. 
> 0. 00 

[■117. 

>ii4. 2 

} 76.7 
V142. 6 


Mgm. 


2 


do 


0. 00 


3 
4 
5 
6 

7 
8 

9 
10 


150 mgm. of NO3 as potassium nitrate . 
do 

150 mgm.^of NO3 as sodium nitrate . . 
do 

150 mgm. of NO3 as calcium nitibte. 
do 

150 mgm . of NO3 as ammonium nitrate 

.... do 


-34-2 
-34-6 

-78.5 
- 8.9 









Table XXV. — Influence of Bacillus radicicola on nitrates in solution giving the quantity 
of nitrogen as ammonia formed 





Treatment (nitrate in loo c. c. of medium). 


Nitrogen as ammonia in loo c. c. of medium. 


Nitro- 


Cul- 
ture 
No. 


Uninoculated. 


Inoculated. 


gen as 
ammo- 
nia 




Found. 


Average. 


Found. 


Average. 


formed. 


I 


None 


Mgm. 
0. 20 
. 10 
. 10 
. 20 
. 20 
. 20 
•40 

-30 
13.90 

13-95 


Mgm. 
} 0-15 

} - 

> . 20 
} 13-92 


Mgm. 
f 0. 10 
\ . 20 
/ . 00 

I • 10 
/ .20 

I -3° 
J -3° 
\ . 10 
f 13. 80 
I 13- 85 


Mgm. 
} 0-15 

} '°^ 
I -25 
> . 20 
} 13-82 


Mgm. 


2 


do 


0. 00 


3 

4 

5 
6 


150 mgm. of NO3 as potassium nitrate . 
do 


— . 10 


150 mgm. of NO3 as sodium nitrate . . 
do 


+-OS 


7 
8 


150 mgm. of NO3 as calcium nitrate. 
do 


-- 15 


9 
10 


150 mgm. of NO3 as ammonium nitrate 
do 


+.10 









Jan. 38, 1918 



Nitrogen- A ssimilating Bacteria 



213 



Table XXVI. — Influence of Bacillus radicicola on nitrates in solution giving the quantity 

of nitrogen fixed 



Cul- 
ture 
No. 



I 
2 
3 

4 
5 
6 

7 
8 

9 
10 



Treatment (nitrate in 100 c. c. of meditun). 



None 

....do 

150 mgm. of NO3 potassium nitrate. . 

. . . .do 

150 mgm. of NO3 sodium nitrate 

....do 

i5omgm. of NO3 calcium nitrate . . . 

. . . .do 

150 mgm. of NO3 ammonium nitrate . 
do 



Total nitrogen in 100 c. c. of medium. 


Uninoculated. 


Inoculated. 


Found. 


Average. 


Found. 


Average. 


Mgm. 


Mgm. 


Mgm. 


Mgm. 


2. 40 
2.50 


} 2.45 


I 3-5° 


I 3- 40 


18.00 
17.90 


} 17- 95 


r 18. 70 
\ 19. 00 


} 18. 85 


16.80 
17. 00 


■ 16. 90 


/ 19- 30 
\ 19. 20 


I 19-25 


14. 00 
13.80 


I 13-90 


f 14. 60 

I 14- 70 


} 14-65 


40. 50 

41. 20 


} 40. 8s 


/ 41- 30 
I 41- 70 


} 41. 50 



Nitro- 
gen 
fixed. 



Mgm.. 
95 



The data in Table XXIV show that a rather large reduction in the 
total nitrate content took place. This reduction varied rather markedly 
among the four dififerent nitrates studied. The greatest reduction oc- 
curred where calcium nitrate was used. Potassium and sodium were 
next in order; the loss was almost the same for both salts. Ammonium 
nitrate was last with but a very small comparative reduction in total 
nitrate. 

The question arises as to whether the nitrate is reduced to nitrite, 
ammonia, or elemental nitrogen or whether the reduction in amount is 
due to a natural assimilation of the nitrate by the organisms. The first 
possibility was precluded when qualitative tests for nitrites were made 
and none found. Table XXV reveals the fact that no ammonia was 
produced. Table XXVI shows no loss in total nitrogen. Therefore it 
seems obvious that reduction in total amount of nitrate present is brought 
about by the assimilation of those compounds by the organisms. 

An inspection of Table XXVI, which gives the results of the total 
nitrogen determinations, shows that a slight fixation of atmospheric 
nitrogen took place. This fixation is entirely possible, as will be shown 
later when the influence of nitrates on the fixation of nitrogen is taken 
up. In the presence of potassium, sodium, and ammonium nitrates the 
amount of nitrogen assimilated is somewhat decreased. But in the 
case of sodium nitrate a large increase in the amount of total nitrogen 
seems to have taken place. This is interesting in the light of results 
to be presented later. 

From the results of the work on the effect of B. radicicola on nitrates 
it may be concluded that the organisms do not reduce the nitrates to 
nitrite or ammonia or elemental nitrogen under aerobic conditions. 



214 Journal of Agricultural Research voi. xii, no. 4 

INFLUENCE OF NITRATES ON THE FIXATION OF ATMOSPHERIC NITROGEN BY BACILLUS 

RADICICOLA 

The ability of B. radicicola to fix atmospheric nitrogen in the absence 
of the host plant has been studied by numerous investigators. From 
the results of their work it seems fairly probable that the legume organ- 
ism can fix nitrogen to a slight extent when growing in a nonsymbiotic 
state. Beijerinck (5) was one of the earliest to make a study of the 
possible fixation of atmospheric nitrogen by B. radicicola under these 
conditions. He found that a small quantity, 0.91 to 1.82 mgm. of nitro- 
gen was fixed per 100 c. c. of the medium. Prasmowski (59, p. 55) and 
Berthelot (6) concluded as a result of their experiments that when the 
organism was grown outside the host plant the gain in nitrogen was 
small. The greatest gain in nitrogen was found by Maze {32) who re- 
ported an increase of 23.4 mgm. of nitrogen per 100 c. c. of the medium in 
16 days. Lewis and Nicholson {30) found by incubating the cultures for 
a considerable length of time that a large increase in fixation occurred. 
Bottomley {8) found that a pure culture of B. radicicola fixed approxi- 
mately I mgm. of nitrogen in 15 days. Fred {17) made a study of the 
possible fixation of nitrogen by the legume organism and found that it 
fixed approximately 1.2 mgm. of nitrogen in 100 c. c. of the medium. 
He found that on agar films a greater fixation occurred than when the 
organisms were grown in a liquid medium. 

A few investigators, however, found that no increase in nitrogen 
occurred when B. radicicola was grown outside the host plant. Frank 
(j6) states that in a nitrogen-free medium the legume organisms did not 
fix enough nitrogen to be accurately measured. Immendorf (25) also 
found no increase in nitrogen when pure cultures of B. radicicola were 
grown in soil containing a nitrogen-free solution. 

It will be seen that the majority of investigators, especially the more 
recent ones, found that a slight amount of atmospheric nitrogen was 
fixed or assimilated by B. radicicola when grown outside the host plant 
and on a medium suitable for its development. 

It has already been shown that nitrates cause an increase in the num- 
ber of B. radicicola when grown in pure culture in sterilized soil. Does 
such an increase in the number of organisms necessarily mean an m- 
creased fixation of nitrogen? Three experiments using agar films were 
carried out in order to determine this point. Erlenmeyer flasks of 
I -liter capacity containing 100 c. c. of mannit agar were used. Before 
the medium solidified, the nitrates were added in the proportions indi- 
cated in Table XXVII. Six flasks for each different quantity of nitrate 
were prepared, except in one case, as shown in Experiment II. The 
flasks were plugged with nonabsorbent cotton and sterilized at 10 pounds' 
pressure for 30 minutes. After cooling, three of each set were inoculated 
with 5 c. c. of a suspension of B. radicicola in sterile distilled water. 
The organisms had been growing on mannit agar at 28° C. for six days. 
The flasks in Experiments I and III (Table XXVII) were incubated at 



Jan. 28, 1918 



Nitrogen- Assimilating Bacteria 



215 



28° C. for three weeks and those in Experiment II for two weeks. The 
moisture lost by evaporation in both inoculated and uninoculated flasks 
was replaced from time to time by the addition of sterile distilled water. 
At the expiration of the incubation period the total nitrogen was deter- 
mined as given under "Methods used in experiments." The results of 
the experiments are given in Table XXVII. 

An inspection of the data reveals the fact that B. radicicola in pure 
culture fixecf a small amount of nitrogen when growing in a nonsymbiotic 
state with no nitrate present. In the presence of nitrates there was an 
increased fixation. Although the increase in total nitrogen is small, 
because of the number of determinations made, it may be considered 
as positive. The potassium and sodium salts seemed to be more effective 
than the calcium nitrate, with one exception (Table XXVII, Experiment 
I). It will be remembered that the latter salt appeared to depress nitro- 
gen fixation by Azotobacter and the two former somewhat to favor it (p. 
194-195). 

TABtE XXVll— Influence of nitrates on the fixation of nitrogen by Bacillus radicicola, 

giving the increase in nitrogen 

EXPERIMENT I 



Culture 
No. 



Treatment (nitrate in 100 c. c. of medium.) 



Total nitrogen in 100 c. c. of medium. 



Uninoculated. 



Foimd. 



None 

....do 

....do 

75 mgm. of NO3 as sodium nitrate . . 

do 

do 

150 mgm. of NO3 as sodium nitrate. 

do 

do 

75 mgm. of NO3 as calcium nitrate . 

do 

do 

150 mgm. of NOjas calcium nitrate . 

do 

do 



Mgm. 

4-5 

4-4 

4-4 

-8.7 

8.7 

8.6 

12.5 

12. 7 



Average. 



8.9 
9.0 

13- I 
13. 2 



Mgm. 
4-45 

8. 70 

12. 60 
8.90 

13. 20 



Inoculated. 



Found. 



Mgm. 



Average. 



Mgm. 
4. 60 

11-75 
14. 70 
12. 40 
14. 10 



Nitrogen 
increase. 



Mgm. 

o. 15 
3-05 



3- SO 
o. 90 



EXPERIMENT II 



None 

....do 

75 mgm. of NO3 as sodium nitrate . . 

do 

150 mgm. of NO3 as sodium nitrate. . 

do 

75 mgm. of NO3 as calcium nitrate . 

do 

150 mgm. of NO3 as calcium nitrate. 
do 



4.90 
4.90 
8. 70 
8.50 

13-30 
13. 00 
II. IS 
II. 10 
14- 70 
(«) 



¥■ 



4.90 
60 



'13- IS 

■II. 125 
^14. 70 




}- 
}<,. 



075 
50 

}i4- 35 
11.65 

15-25 



o- 175 

0. 90 

1. 20 
o. 525 
0-550 



a Lost by breakage during sterilization. 



2l6 



Journal of Agricultural Research vo1.xii.no. 4 



Table XXVII. — Influence of nitrates on the fixation of nitrogen by Bacillus radicicola, 
giving the increase in nitrogen — Continued 



EXPERIMENT III 



Culture 
No. 



Treatment (nitrate in loo c. c. of medium): 



9 
10 
II 
12 
13 
14 
15 
16 

17 
18 

19 
20 



None 

...do 

....do 

75 mgm. of NO3 as potassium ni- 
trate 

....do 

....do _. 

150 mgm. of NO3 as potassium ni- 
trate 

...do 

....do 

75 mgm. of NO3 as sodium nitrate 

....do 

....do 

150 mgm. of NOgas sodium nitrate 

....do 

....do 

75 mgm. of NO3 as calcium nitrate 

....do 

....do 

150 mgm. of NOjas calcium nitrate 

....do, 

....do 



Total nitrogen in 


ICX5 c. c. of medium. 


Uninoculated. 


Inoculated. 


Found. 


Average. 


Found. 


Average. 


Mgm. 


Mgm. 


Mgm. 


Mgm. 


5.10 




\ 5-50 


1 


5. 10 


1 5-07 


5- 40 


[ 5-50 


5. CO 




I 5-45 


J 


9-35 


) 


f 10.85 


1 


9-5" 


[ 9-37 


{ 10. 90 


\ 10. 90 


9-25 


J 


I 10.95 


J 


14.50 




[ 15-65 




14. 20 


[14. 28 


\ 15-30 


1 5-. 45 


14-15 




I 15-40 




8. 50 




[ 9-85 




8.30 


8.38 


\ 9-90 


9-83 


8.35 




I 9- 70 




12.3s 




f 12.95 




12. 40 


[12.33 


j 13-10 


[ 13-03 


12. 20 




I 13-05 




8-95 




f 985 




9. 10 


r 9- °i 


\ 9- 90 


[ 9-93 


9. 00 




I 10.05 




13.90 




f 14.40 




13.80 


13.80 


14-50 


[ 14- 42 


13.70 




\ 14-35 





Nitrogen 
inca-ease. 



Mgm,. 
0-43 

1-53 
I. 17 

1-45 
o. 70 
o. 92 
o. 62 



It has been shown that, when nitrates are added in varying quantities 
to sterilized soil, the number of B. radicicola are increased. Provided the 
the organism can fix a small amount of nitrogen in the absence of nitrate 
nitrogen, is it not possible that this increase in nitrogen fixation may be 
due merely to the increase in the number of cells ? It seems that this is 
true according to the results in Table XXVII. It appears probable that 
the increase in nitrogen fixed in the presence of nitrates is very likely 
because of an increase in the number of bacterial cells and not to any 
physiological change brought about in the organism itself. 

There was a marked increase in bacterial growth on the media con- 
taining the nitrate compared with the same media free from nitrate. 
The growth on the latter medium exhibited a normal, whitish watery 
appearance, characteristic of this organism. On the cultures containing 
nitrates a much more profuse growth occurred. In, many instances a 
pinkish tint was observed. This pigment production was especially 
marked in the case of the culture containing the sodium salt. After the 
first experiment had been completed, it was thought that possibly this 
pigmentation was due to an impurity in the culture. Therefore the two 
remaining experiments were made, using a subculture from the original. 



Jan. 28. i9i8 Nitrogen-Assimilating Bacteria 217 

This culture was plated three times, each plating being made from a well- 
isolated colony. The final subculture was taken from a similar well- 
isolated colony. However, pigment formation in the presence of nitrate 
persisted in the two final experiments, showing clearly that some reaction 
took place between the nitrate and the organism grown on the medium. 
It is of interest to note that the pigment formation in the presence of 
nitrate was observed in later work where the influence of nitrates on 
nodule formation was investigated. Prucha (41) found that on agar 
slopes of medium containing 0.5 per cent of potassium or calcium 
nitrate, the growth of B. radicicola became opaque and that an iridescent 
tint was produced. 

Although the results of these experiments may vary somewhat among 
themselves, taken as a whole it appears evident that B. radicicola may 
fix a small amount of atmospheric nitrogen when grown without the 
host plant and on a suitable medium. The addition of various amounts 
of nitrates as indicated increased somewhat the amount of nitrogen 
assimilated by B. radicicola. 

INFLUENCE OP NITRATES ON THE PRODUCTION OP GUM BY BACILLUS RADICICOLA , 

Since nitrates, especially in smaller amounts, cause an increase in the 
number of B. radicicola in pure culture, it was thought advisable to 
determine what influence these salts have on the production of gum. In 
culture media favorable to the growth of B. radicicola these bacteria will 
produce a gelatinous substance which is readily precipitated with 95 per 
cent alcohol or acetone. Upon the addition of either of these precipi- 
tants a fairly heavy, water-white, frothy gelatinous mass is formed 
which soon rises to the surface of the liquid. Upon standing, this mass 
contracts somewhat, and portions of it may fall to the bottom of the 
liquid from which it has been precipitated. 

Chemical analyses, according to Buchanan (10), have shown that this 
gum is a carbohydrate. Upon hydrolysis with 2 per cent sulphuric acid 
and 15 pounds' pressure for one hour, Fehling's solution is reduced, 
showing the presence of a sugar. The gum does not give proteid reac- 
tions with the Millon, biuret, or xanthoproteic tests. Hence, the gum 
is not protein in character; nor does it contain nitrogen in the combined 
form. Clearly it is a nonnitrogenous body. 

In the experiment undertaken to determine whether nitrates influence 
the formation of gum only relative difi"erences are noted. No attempt 
was made to obtain quantitative results. 

Erlenmeyer flasks of i -liter capacity containing 200 c. c. of mannit 
solution were used. The cultures contained various quantities of nitrate 
as indicated in Table XXVIII. Triplicate flasks for each amount of 
nitrate were prepared. In this table these three flasks are represented 
as "a," "b," and "c." After sterilization at 15 pounds' pressure for 25 
minutes the flasks were cooled and inoculated with 5 c. c. of a suspension 



2l8 



Journal of Agricultural Research 



Vol. XII, No. 4 



of B. radicicola in sterile distilled water. The cultures were then 
incubated at room temperature (approximately 25° C.) for eight weeks. 

At the expiration of the incubation period the contents of the flasks 
were poured into hydrometer cylinders of equal depth and diameter. 
One hundred and fifty c. c. of acetone were added to precipitate the gum. 
After careful shaking, the cylinders were covered with inverted petri 
dishes to prevent evaporation. At the end of 24 hours the amount ol 
gum precipitated was observed. The relative amounts are recorded in 
Table XXVIII. 

Table XXVIII. — Influence of nitrates on the production of gum by Bacillus radicicola 



Cul- 


Treatment (nitrate in loo c. c. of medium). 


Relative production of gimi— precipitated by 
acetone. 


No. 


Flask a. 


Flask b. 


Flask c. 




None 


Large 

Very large . 

Large 

Very large . 
Laree 


Large 

Very large . 

Large 

...do 

...do 


Large. 


2 

3 
4 

5 


75 mgm. of NO3 as potassium nitrate . 
450 mgm. of NO3 as potassium nitrate . 
75 mgm. of NO3 as sodium nitrate .... 
450 mgm. of NO3 as sodium nitrate. . . 
7^ msrm of NO-> as calcium nitrate 


Very large. 
Large. 
Very large. 
Large. 
Do. 


. do 


...do 


7 


4t;o mgm of NO3 as calcium nitrate. . 


...do 


C nsider- 
able. 


Con sider- 






able. 



From the results it is certain that the nitrates, especially in the smaller 
of the two concentrates, caused a very considerable increase in the amount 
of gum produced by B. radicicola. The nitrates of potassium and sodium 
caused a production of more gum than did the calcium salt. It will be 
remembered that in the experiments where the influence of nitrates on 
the fixation of atmospheric nitrogen by B. radicicola was studied, less 
nitrogen was fixed in the presence of calcium nitrate than in the pres- 
ence of the other two salts. Here again the greater stimulative action 
of potassium and sodium nitrates is emphasized. 

Buchanan in his investigations on the formation of gum by B. radici- 
cola has found that varying amounts of potassium nitrate in a 2 per 
cent saccharose solution or in a 2 per cent saccharose-clover-extract 
solution caused a slight increase in growth and in gum production. 

It seems probable that the increased gum production in the nitrate 
cultures is caused not only by an increase in bacterial cells but also 
perhaps by an increased stimulation in the formation of gum by the cells 
themselves. The relative increase in the amount of gum produced in 
the presence of nitrates seems to be greater than the actual increase in 
number of organisms brought about by the stimulating effect of the 
nitrate. In the latter instance this stimulating effect has been deter- 
mined in soil cultures only and so a fair basis of comparison can not be 



Jan. 28, i9i8 Nttrogen-Assimilating Bocterta 219 

found. Had the influence of nitrates on the growth and reproduction 
of B. radicicola been determined in liquid culture, as well as in soil 
cultures, then a comparison could have been made. Furthermore, the 
divergencies in the time element, eight weeks' incubation in the liquid 
cultures and three weeks in the soil cultures, are such as to render futile 
any attempt at correlation. It may be that the large formation of gum 
was due to the prolonged incubation. A shorter period of three weeks 
undoubtedly would show a relatively smaller amount of gum produced 
as a result of the presence of the nitrate. 

However, from the results of the experiment it is certain that potas- 
sium, sodium, and -calcium nitrate influence the formation of gum by B. 
radicicola. The three nitrates studied caused a large increase in the 
amount of gum obtained by precipitation with acetone. Calcium nitrate 
caused the least stimulation, but the difference was not large. 

INFLUENCE OF NITRATES ON NODULE FORMATION 

The results of numerous investigations have shown that nitrates 
retard and oftentimes entirely prevent the formation of nodules on 
leguminous plants when grown in soil or liquid cultures. Vines (45), 
working with the horse bean, found that the use of large amounts of 
nitrate iii the form of potassium nitrate retarded nodule formation. He 
concluded that a decrease in the amount of nitrates meant an increase 
in the number of nodules. Vv'oods {4S) found that leguminous plants 
assimilated more nitrogen when they were grown in the absence of 
potassium and calcium nitrate than* when thus supplied. His results 
seem to indicate that nodule development was retarded somewhat by 
these salts. Similar results were obtained by Frank (z6). Nobbe and 
Richter (57) in 1902 grew soybeans in a rich garden soil and found upon 
inoculation that a gain of 74.7 per cent of nitrogen occurred. However, 
upon the addition of nitrates this gain was considerably reduced, the extent 
of the reduction corresponding to the amount of nitrate added. About 
this same time, Wohltmann and Bergene (47) using many diflierent 
types of soils, found that nodules were not formed on the roots of peas 
when ammonium nitrate was added, Creydt {12) found that sodium 
nitrate retarded nodule formation on yellow lupines when these legumes 
were grown in soil, Fred and Graul {18) found that very small amounts 
of nitrates did not appreciably decrease nodule formation, but that 
larger amounts proved detrimental and finally prohibited entirely the 
development of nodules. 

The presence of nitrates in culture solutions has also been found to 
reduce and oftentimes to inhibit the formation of nodules on leguminous 
plants. Marchal {31) concluded that alkaline nitrates in concentrations 
of I to 10,000 in liquid cultures prevented the formation of nodules on 
peas. Flamand {15) grew vetch and beans in a nutrient solution and 



220 Journal of Agricultural Research voi. xii, no. 4 

found that nitrates in the following amounts prevented nodule forma- 
tions: potassium nitrate, i to 10,000, sodium nitrate i to 2,000, ammo- 
nium nitrate i to 2,000, and calcium nitrate i to 2,000 and i to 10.000. 
Hiltner's {24) experiments showed that 5 mgm. of nitrogen as potassium 
nitrate per liter prevented nodule formation on peas. 

In contrast to these experiments Bassler (j) claimed that results 
obtained from his work indicated that no effect was noticed by adding 
nitrates to lupines growing in quartz sand. 

The question naturally arises whether this condition is due to the 
weakening of the organism brought about by growth in a nitrated 
environment and to a consequent impairment or entire loss of its infect- 
ing power, or whether it is caused by some interreaction between the 
salt and the plant root, tending to increase the latter's resistance to 
the attack of this particular organism. 

INFLUENCE OF NITRATES ON THE INFECTING POWER OF BACILLUS RADICICOLA 

Some investigations have been carried out to determine what effect 
nitrates have on the legume organisms themselves. Wilson {46) showed 
that although nitrates inhibit the formation of nodules, the organisms 
capable of producing nodules did not lose their vitality or nodule-pro- 
ducing power when grown in the presence of nitrates. The results of 
Prucha {41) are in accord with those of Wilson. He found that B. radi- 
cicola does not seem to lose its infecting power when grown on media 
containing nitrate. During the course of his work he found that potas- 
sium and sodium nitrates inhibited the formation of nodules. Further 
evidence that the organisms appear to retain their vitality in the pres- 
ence of nitrates is produced by the results of Maze (jj, p. 15-iy), who 
showed that legume bacteria were able to fix a slight amount of nitrogen 
when grown in a soil extract solution containing i per cent sodium nitrate. 
Herke {22) states that potassium nitrate favors the growth of nodule 
bacteria. 

However, other investigators state that nitrates have a harmful 
effect on B. radicicola. Laurent (29, p. 134) found that legume organ- 
isms failed to grow in a pea or lupine decoction containing nitrate in the 
form of potassium and sodium salts in amounts equivalent to i to 500 
and I to 1,000. Moore (55) in his experiments demonstrated that nitrates 
at I to 10,000 were sufficient to prevent nodule formation. He states 
that B. radicicola loses its power of infection when grown in a medium 
containing nitrates. 

From the results cited it can be seen that there is some disagreement 
as to the influence exerted by nitrates on B. rad'i/:icola. In some cases 
the organism seems to retain its vitality in the presence of nitrates, 
while in others it appears to have become weakened. It must be ad- 



Jan. 28, 1918 



Nitrogen- Assimilating Bacteria 



221 



mitted, however, that the evidence seems to favor the former contention — 
namely, that nitrates do not cause the bacteria to lose their nodule- 
producing power. 

In order to determine whether or not nitrates weaken these organisms, 
the following experiments were made: Slopes of mannit agar (in test 
tubes) containing various amountsof sodium and calcium nitratesas indi- 
cated in Table XXIX were inoculated with B. radicicola. These cul- 
tures were incubated at 28° C. for one week, when transfers were made 
to fresh nitrate media and incubated at 28° C. for another week. At 
the expiration of this time, three 4-day-old seedlings of alfalfa were 
inoculated with three drops of a suspension of the organism in 5 c. c, 
of sterile distilled water. The same slope cultures were incubated at 
28° C. and used for all subsequent inoculations in this experiment. 
The inoculated seedlings were placed in the greenhouse under cheese- 
cloth covering. The temperature here during the daytime averaged 
approximately 30° C. The seedlings were examined for the first appear- 
ance of nodules and in no case did they appear before 18 to 20 days. 
A total count of nodules on all plants was made at the end of 45 days. 
Three subsequent inoculations were made under the same conditions. 
In this way organisms in contact with nitrate for varying lengths of 
time could be used. The results of the inoculation experiments are 
given in Table XXIX. 



Table XX-IX. — Influence of nitrates on the infecting power of Bacillus radicicola 



Cul- 


Treatment (nitrate in loo c. c. of medium). 


Number of nodules after 43 days. 


ture 
No. 


Inoculated 
June 3. 


Inoculated 
June 15. 


Inoculated 
July II. 


Inoculated 
July 17. 


I 


None 


5 
5 
7 
4 
5 
6 
2 
7 
4 
9 
5 
7 
5 
6 




4 
4 
6 

5 
8 

5 
4 
8 

4 
4 
5 
5 
7 
8 




7 
5 

15 
3 
4 

5 
6 

7 
9 
8 
II 
9 
4 
6 




3 

5 

5 

II 
8 
7 

3 
8 


2 

3 
4 
5 
6 

7 
8 


15 mgm. of NO3 as sodium nitrate . . . 
37 mgm. of NO3 as sodium nitrate . . . 
75 mgm. of NO3 as sodium nitrate . . . 
150 mgm. of NO3 as sodium nitrate . . 
225 mgm. of NO3 as sodium nitrate . . 
450 mgm. of NO3 as sodium nitrate . . 
None 


9 
10 
II 
12 

13 
14 
15 
i6 


15 mgm. of NO3 as calcium nitrate . . 
37 mgm. of NO3 as calcium nitrate. . . 
75 mgm. of NO3 as calcium nitrate . . 
150 mgm. of NO3 as calcium nitrate. . 
225 mgm. of NO3 as calcium nitrate . 
450 mgm. of NO3 as calcium nitrate . 
Uninoculated 


4 
6 
8 
9 
3 
3 


do 












From the results given in Table XXIX it is very evident that under 
the conditions of the experiment the legume organisms did not lose 
their power of producing nodules when grown on a medium containing 



222 Journal of Agricultural Research voi. xii, no. 4 

varying amounts of sodium and calcium nitrates. The numbers of 
nodules produced on the alfalfa plants by organisms grown on media 
containing nitrate do not vary widely from those on the plants inocu- 
lated with organisms grown on media containing no nitrate. Not only 
did the organisms fail to lose their nodule-producing power, but from 
all appearances their infecting power did not seem to be materially 
weakened. 

It therefore seems apparent that an explanation for the failure of 
nodules to develop on leguminous plants in the presence of nitrates is 
not found in the theory that the organisms producing these nodules are 
weakened when grown in the presence of nitrates. 

INFLUENCE OF NITRATES ON ALFALFA ROOTS AND NODULE FORMATION 

The next step taken would naturally be in the direction of a study of 
the influence of the nitrates on the plant roots themselves in order to 
determine whether or not they thus are made more resistant to the 
attack of these organisms. 

A review of the literature shows that almost nothing has been done 
touching this phase of the question. Wilson {46) y studying the effect of 
certain salts on nodule production, states that possibly the salt has some 
effect on the root, making it resistant to the attack of the organism. 
Maze {33, p. 15-17), who also concluded that nitrates did not cause B. 
radicicola to lose its infecting power, says that nodules do not develop 
on roots of legumes when nitrates are present because the carbohydrate 
in the roots is changed into protein material by the absorption of the 
nitrate. 

Alfalfa seedlings (Medicago saliva) growing in soft agar containing 
potassium, sodium, and calcium nitrates, as indicated in Table XXX, 
were used in this study. Quadruplicate tubes were prepared for each 
amount of nitrate. The higher concentrations of the nitrate were not 
used, since it was found that germination and subsequent growth were 
considerably impaired in the presence of such large amounts. The tubes 
with the mannit agar and nitrate were sterilized at 1 5 pounds' pressure 
for 30 minutes. These were cooled and sterilized alfalfa seeds planted 
as given under "Methods used in experiments." The tubes were 
then placed in the greenhouse under cheesecloth covering and the seeds 
allowed to germinate. Germination took place in all instances, although 
it was retarded somewhat by the presence of the nitrate. At the end of 
five days the first tube of each set was inoculated with three drops of a 
suspension of B. radicicola in sterile distilled water. Subsequent inocu- 
lations were made as indicated in Table XXX. These were made at 
different intervals in order to allow the roots of the seedlings to remain 
for a longer time in contact with the media. It was hoped that in this 
way an idea might be obtained as to the time when the root first became 
resistant. The results are given in Table XXX. 



Jan. 28, 1918 



Nitrogen- A ssimilating Bacteria 



223 



Table XXX.— Influence of nitrates on alfalfa roots and nodule formation 



Culture 
No. 



Total number of nodules in each tube of 
seedlings inoculated after — 



Treatment (nitrate in 100 c. c. of medium). 



None V 

10 mgm. of NO3 as potassium nitrate . 
25 mgm. of NO3 as potassium nitrate . 
50 mgm. of NO3 as potassium nitrate . 
100 mgm. of NO3 as potassium nitrate 
150 mgm. of NO3 as potassium nitrate 
ID mgm. of NO3 as sodium nitrate . . . . 
25 mgm. of NO3 as sodium nitrate . . . . 
50 mgm. of NO3 as sodium nitrate . . . . 
100 mgm. of NO3 as sodium nitrate . . . 
150 mgm. of NO3 as sodium nitrate . . . 
10 mgm. of NO3 as calcium nitrate . . . 
25 mgm. of NO3 as calcium nitrate . . . 
50 mgm. of NO3 as calcium nitrate . . . 
100 mgm. of NO3 as calcium nitrate . 
150 mgm. of NO3 as calcium nitrate . . 



5 days' 
growth. 



10 days' 
growth. 






l^) 



18 days' 
growth. 



Q') 



(^) 



22 days' 
growth. 



(«) 



a Fungus contamination. 



& Plant died after few days' growth. 



It will be seen that in a few instances where a high concentration of 
nitrates occurred the development of the seedlings subsequent to ger- 
mination ceased. This condition may have been due to too high a con- 
centration of soluble salts or to inferior seed. However, losses were not 
sufficiently serious materially to affect the outcome of the experiment. 

In all cases the seedlings grown in agar without nitrate produced 
nodules when inoculated with B. radicicola. A few nodules appeared on 
seedhngs in cultures containing the lowest concentration of all three 
nitrates. The number of nodules in these cases was less than in the 
control cultures. No nodules whatever developed in any concentration 
above lo mgm. of nitrate in lOo c. c. of medium. Under normal condi- 
tions in test-tube cultures the nodules make their appearance at about 
18 to 20 days after inoculation. The incubation of all cultures was 
extended 40 days after inoculation in order to make certain that no 
further nodule development would take place. 

The nonproduction of nodules was not due to the failure of the inoc- 
ulum. In all cases an excellent inoculum growth was obtained, espe- 
cially in the case where nitrate was present in the medium. Indeed, it 
was so luxuriant that in many cases the organism grew in considerable 
quantity far down into the root zone. In many cases where nitrates 
were present the same pink coloration was produced that was discussed 
under another caption, on page 216. 

As has been already stated, seedlings of varying ages were moculated 
for the reason that it was thought that a more or less prolonged contact 
of the roots with the nitrate in the medium might serve as an mdex to 



224 Journal of Agricultural Research voi. xii. No. 4 

the time in the growth of the seedling when permanent resistance to 
attack of the organisms was established. The results obtained do not 
seem to indicate that seedling roots 18 to 20 days' old are any more 
resistant to the attack of the organisms than are those that are younger. 
Evidently if any reaction takes place between the nitrate and the plant 
root it occurs during the very early stages in the development of the 
plant. 

These results seem to point to the conclusions that the nonformation 
of nodules in the presence of nitrates is due not to a weakening of the 
vitality of the organism, but to some reaction between the plant root 
and nitrate. One naturally queries whether the plant root cells are 
made more resistant to the bacteria seeking to gain entrance there or 
whether the naturally occurring carbohydrate food supply to be used 
by the organisms after gaining entrance is diminished by its conversion 
into protein material by the absorption of nitrate? Further studies 
were not made in an endeavor to solve this question. 

INFLUENCE OP NITRATES IN SOIL ON ALFALFA NODULES AND ON THE REFORMATION 

OF NODULES 

Additional studies were made with nitrates in relation to their influ- 
ence on nodules already formed and on the redevelopment of nodules 
once removed from alfalfa plants. The experiments were carried out 
in an endeavor to determine whether nitrates prevented an increase in 
the number of nodules on plants possessing nodules and whether they 
prevented the reformation of nodules when removed. Experiments 
revealed clearly that removed nodules were replaced by new ones pro- 
vided the plant was carefully replaced in the soil (soil with normal low 
nitrate content) and the proper amount of moisture maintained. 

In these experiments i -gallon earthenware jars were used. These 
were filled to within an inch of the top with 1,800 gm. of soil of a low 
nitrate content. Different amounts of the nitrates to be studied were 
added in the quantities indicated in Table XXXI. Concentrations of 
100 and 300 mgm. of nitrate in 100 gm. of soil were also used, but the 
transplanted alfalfa seedlings were unable to withstand such excessive 
concentration, with the result that all died within a week or ten days 
after transplanting. Quadruplicate pots were prepared for each con- 
centration of nitrate. The nitrates in solution were mixed with the 
proper amount of distilled water which, when added to the pots, brought 
the moisture content to approximately 20 per cent. The pots were then 
allowed to remain undisturbed for one day at room temperature to 
allow the water containing the nitrate to become well diffused through- 
out the soil mass. Into two pots of each set were transplanted young 
alfalfa plants from which the nodules had been removed. The two 
remaining pots contained transplanted alfalfa plants with the nodules 
left on and their location noted. The plants used in this experiment 



Jan. 38, J918 



Nitrogen- A ssimilating Bacteria 



225 



were removed from an alfalfa plot, the soil of which was a sandy loam. 
Previous to transplanting the roots of the young plants were carefully 
washed in running water and immediately transplanted. The pots 
were kept well watered, and after two or three days they were removed 
to the greenhouse. Here they were watered when necessary. Trans- 
plantations were made on the 27th of June and the experiment termi- 
nated on the 3d of August. The plants were removed from the pots, 
the roots carefully washed and examined for the presence of nodules. 
The results are presented in Table XXXI. 



Table XXXI. 



-Influence of nitrates in soil on alfalfa nodules and on the reformation of 
nodtdes 



Pot No. 



Al. 
A2. 

A3- 
A4. 
B I. 

B2. 

B3- 
B4. 
Ci. 
C2. 
C3- 
C4. 
D I. 
D2. 
D3- 
D4. 
Bi. 
E2. 

§3- 
E4. 
Fi. 
F2. 
F3. 
F4. 
Gi. 
G2. 
G3- 
G4. 



Nitrate ia loo gm. of dry soil. 



None 

....do 

....do 

....do 

25 mgm, of NO3 as potassium nitrate 
do 

....do 

...do 

50 mgm. of NO3 as potassium nitrate. 

... .do 

do 

do 

25 mgm. of NO3 as sodium nitrate . . . 

do 

do 

do 

50 mgm. of NO3 as sodium nitrated . . 

do 

do 

do 

25 mgm. of NO3 as calcium nitrate . . 

do 

do 

do 

50 mgm. of NO3 as calcium nitrate . . 

do 

do 

do 



Treatment of 
nodules. 



Removed 

....do 

Not removed . 

do 

Removed 

do 

Not removed . 

do 

Removed . . . . 

do 

Not removed . 

do 

Removed.. .. 

do 

Not removed . 

do 

Removed.. .. 

do 

Not removed . 

do 

Removed.. .. 

do 

Not removed . 

do 

Removed.. .. 

do 

Not removed . 
do 



Number of 
nodules — 



At 
bedn- 
ning. 



At 
end. 



(a) 



(«) 



" Plants died. 

It will be seen in the control pots, where no nitrate was present (ex- 
cept the small amount normally present in the soil at the beginning of 
the experiment), that if the nodules were removed, new ones formed. 
The location of the nodules before their removal was noted, and the 
new ones were found to occupy the same place. However, when nitrates 
were added to the soil no new nodules were formed. This statement 
holds true for both concentrations of all three salts in all experiments. 



2 26 Journal of Agricultural Research voi. xii, N0.4 

Some interesting results were obtained where the nodules were not 
removed. In the control pots an increase in nodule formation took place. 
It can not be stated definitely whether the new nodules appeared as 
a result of inoculation from the soil or whether the organisms had already 
gained entrance to the roots before the plants were removed from the 
field soil previous to transplanting. Nevertheless, it is shown that the 
number of nodules increased as compared with the number present at 
the time of transplanting. But where nitrates were added a reduction 
in number occurred rather regularly throughout all the pots. In two 
instances the number remained constant, in 10 it was reduced, and in none 
was it increased. The calcium salt appeared to effect the least reduction 
in number of nodules. Conclusions concerning the comparative in- 
fluence of the three salts in this regard cd,n not be drawn because of the 
small number of determinations made. It is sufficient to note that 
nitrates present in amounts equal to 25 and 50 mgm. of nitrate in 100 
gm. of soil did not permit an increase in number of nodules, but rather 
caused a decrease. 

The conclusions drawn from the experiments relative to the influence 
of nitrates on nodule formation are: (a) the presence of nitrates is 
detrimental to the formation of nodules by alfalfa; (b) the nonformation 
of nodules is not due to a weakening of B. radicicola when grown in the 
presence of nitrates; (c) some reaction takes place between the nitrates 
and the plant root, thus preventing nodule formation; (d) nitrates in the 
soil prevent, the re-formation of nodules once removed and also cause 
a decrease in the number of those already present. 

SUMMARY 

(i) Small quantities of potassium, sodium, and calcium nitrates 
caused a great increase in the number of Azotobacter in sterilized soil.. 
Ammonium nitrate in the same quantities caused a less marked in- 
crease. Higher concentrations were not so favorable to the growth 
of the organisms. 

(2) Potassium and sodium nitrates in the concentrations studied 
caused an increase in the amount of nitrogen assimilated by Azoto- 
bacter on agar films. Calcium nitrate in the same amounts brought 
about a decrease in the amount of nitrogen fixed to a point even below 
that representing the amount assimilated in the absence of nitrates. In 
soil cultures nitrates of sodium and calcium caused an increase in total ni- 
trogen, which was more marked in the unsterilized cultures than in those 
cultures sterilized and inoculated with a pure culture of Azotobacter. 
However, the increase in total nitrogen is not commensurate Vv^ith the 
increase in the number of Azotobacter noted under the same conditions. 

(3) Under aerobic conditions Azotobacter in liquid cultures reduced 
nitrate to nitrite, but not to ammonia. More atmospheric nitrogen was 
assimilated in the presence of nitrate than in the absence of this salt. 



Jan. 28, i9i8 NUrogen-Asshnilating Bacteria 227 

(4) Pigmentation occurred when potassium and sodium nitrates, 
and especially calcium nitrate, were used with Azotobacter, the colora- 
tion increasing with the concentration of the salt. This effect was more 
marked in Azotobacter strains which produce little or no pigment in the 
absence of nitrates. 

(5) All three nitrates studied caused an increase in the number and 
size of volutin bodies in Azotobacter cells. From all appearances 
these salts also tended to hasten the development of these bodies. 

(6) The number of Bacillus radicicola in sterilized soil was increased 
by the addition of small quantities of potassium, sodium, ammonium, 
and calcium nitrates. This increase was not so marked as in the Azoto- 
bacter cultures. B. radicicola appeared to be much more resistant to 
higher concentrations of nitrates than Azotobacter. 

(7) B. radicicola under aerobic conditions did not reduce nitrates 
in solution to nitrite, ammonia, or elemental nitrogen. The presence 
of nitrates did not materially influence the small amount of atmos- 
pheric nitrogen fixed under these conditions. 

(8) When grown on agar films, B. radicicola fixed a small amount 
of nitrogen, varying from 0.15 to 0.43 mgm. of nitrogen in 100 c. c. of 
the medium. The addition of various amounts of potassium, sodium, 
and calcium nitrates increased to a slight extent the amount of nitrogen 
assimilated. 

(9) In hquid cultures all three nitrates caused a large increase in the 
amount of gum obtained by precipitation with acetone. 

(10) The presence of large amounts of potassium, sodium, and cal- 
cium nitrates proved detrimental to the formation of nodules on alfalfa. 
B. radicicola did not appear to lose its infecting power when grown on 
media containing varying amounts of sodium and calcium nitrates. 
Alfalfa seedUngs grown in the presence of large amounts of nitrate did 
not produce nodules when inoculated with a viable culture of B. radicicola. 
Nitrates in soil cultures prevented the re-formation of nodules once 
removed and also caused a decrease in the number of nodules already 
present. 

LITERATURE CITED 
(i) Bassler, p. 

1895. SANDKULTURVERSUCHE UBER DIE STICKSTOFPASSIMILATION DER GELBEN 
LUPINE IM STERILISIERTEN UND GEIMPFTEN BODE.N BEI DARGEBOT 
WECHSELNDER MENGEN VON SALPETERSAUREN SALZEN. In JahresbcT. 

Agr. Chem., n. F., Bd. 18 (Jahrg. 38), p. 131. 

(2) Beijerinck, M. W. 

1888. DIE BACTERiEN DER P.VPILIONACEEN-KNGLLCHEM. In Bot. Ztg., Jahrg. 
46, No. 48, p. 758-771. (Continued article.) 

(3) — 

189 1. OVER OPHOOPING VAN ATMOSPHERISCHE STICKSTOP IN CULTUREN VAN 

BACILLUS RADICICOLA. In Vcfslag. en Meded. K. Akad. Wetensch. 
[Amsterdam], r. 3, deel 8, p. 460-475. 



228 Journal of Agricultural Research voi. xii, No. 4 

(4) Beijerinck, M. W. 

1901. UEBER OLiGONiTROPHiLE MiKROBEN. In Centbl. Bakt. [etc.], Abt. 2, 

Bd. 7, No. 16, p. 561-582, I pi. 

(5) -^ and DeldEN, A. van. 

1902. UEBER DIE ASSIMILATION DES FREIEN STICKSTOFFS DURCH BAKTERIEN. In 

Centbl. Bakt. [etc.], Abt. 2, Bd. 9, no. 1/2, p. 3-43. 

(6) BerTHELOT, M. p. E. 

1893. RECHERCHES NOUVELLES SUR LES MICRORGANISMES FIXATEURS DE t' 

AZOTE. In Compt. Rend. Acad. S'ci. [Paris], t. 116, no. 17, p. 842-849. 

(7) BoNAZZi, Augusto. 

I915. CYTOLOGICAL STUDIES OF A^OTOBACTER CHROOCOCCUM. In Jour. Agr. 

Research, v. 4, no. 3, p. 225-239. Literature cited, p. 238-239. 

(8) BOTTOMLEY, W. B. 

1909. SOME EFFECTS OF NITROGEN-FIXING BACTERIA ON THE GROWTH OP NON- 
LEGUMINOUS PLANTS. In Proc. Roy. Soc. London, s. B, v. 81, no. 
548, p. 287-289. 

(9) Bredemann, G. 

1909. BACILLUS AMYLOBACTER A. M. ET BREDEMANN IN MORPHOLOGISCHER, 
PHYSIOLOGISCHER UND SYSTEMATISCHER BEZIEHUNG. In Centbl. Bakt 
[etc.], Abt. 2, Bd. 23, no. 14/20, p. 385-568, 13 fig., 2 pi. Literatur- 
verzeichnis, p. 559-566. 

(10) Buchanan, R. E. 

1909. THE GUM PRODUCED BY BACiLUS RADicicoLA. In Centbl. Bakt. [etc.], 

Abt. 2, Bd. 22, No. 11/13, p. 371-396. Citations, p. 395-396. 

(11) Chalmot, G. de 

1894. THE influence of nitrates on GERMINATING SEEDS. In Agr. Science, 

V. 8, no. lo/ii, p. 463-465. 

(12) Creydt, Bodo. 

1915. Ul^TERSUCHUNGEN IJBER DIE KALKEMPFINDLICHKEIT DER LUPINE UND 

IHRE BEKAMPFUNG. In JouT. Laudw., Bd. 63, Heft 2, p. 125-191, 6 

pi. 

(is) Drabble, Eric, and Scott, Daisy G. 

1907. ON THE EFFECT OF ACIDS, ALKALIS, AND NEUTRAL SALTS ON THE FER- 
MENTATIVE ACTIVITY AND ON THE RATE OF MULTIPLICATION OF YEAST 

CELLS. In Biochem. Jour., v. 2, no. 7/8, p. 340-349, i fig. Literature 

P- 349- 

(14) Fernbach, a., and Lanzenberg, A. 

1910. DE L'aCTION DES NITRATES DANS LA FERMENTATION ALCOOLIQUE. In 

Compt. Rend. Acad. Sci. [Paris], t. 151, no. 17, p. 726-729. 

(15) Flamand, Henri. 

1905. UBER DEN EINFLUSS DER ERNAHRUNG AUF DIE ENTWICKLUNG DER 

KNOLLCHEN DER LEGUMiNOSEN. In Centbl. Agr. Chem., Jahrg. 34, 
Heft II, p. 738-740. 

(16) Frank, B. 

1892. DIE ASSIMILATION FREIEN STICKSTOFFS BEI DEN PFLANZEN IN IHRER 
ABHANGIGKEIT VON SPECIES, VON ERNAHRUNGSVERHALTNISSEN UND 

VON BODENARTEN. In Landw. Jahrb., Bd. 21, p. 1-44. 

(17) Fred, E. B. 

1913. A PHYSIOLOGICAL STUDY OF THE LEGUME BACTERIA. In Va. Agr. Exp. 

Sta., Ann. Rpt., 1911/12, p. 145-173, fig. 34. Literature, p. 172-173. 
(18) and Graul, E. J. 

1916. THE EFFECT OF SOLUBLE NITROGENOUS SALTS ON NODULE FORMATION. 

In Jour. Amer. Soc. Agron., v. 8, no. 5, p. 316-328. Literature cited, 
p. 327-328. 



Jan. 28, i9i8 Nitrogen-Assimilatiug Bacteria 229 

(19) Greaves, J. E. 

I916. THE INFLUENCE OF SALTS ON THE BACTERIAL ACTIVITIES OF THE SOIL. In 

Soil Science, v. 2, no. 5, p. 443-480, 4 fig. 

(20) Hanzawa, J. 

I914. EINIGE BEOBACIITUNGEN UBER STICKSTOFF-BINDUNG DURCII AZOTOBAC- 
TER IN STICKSTOFFARMEN UND IN STICKSTOFFREICHEN SUBSTRATEN. 

In Centbl. Bakt. [etc.], Abt. 2, Bd. 41, No. 18/23, p. 573-576- 

(21) HELLRIEGEL, H., and Wilfarth, H. 

1888. UNTERSUCHUNGEN UBER DIE STICKSTOFFNAHRUNG DER GRAMINEEN UND 

LEGUMiNOSEN. In Ztsclir. Ver. Riibenzuckerindus., Beilageheft, 
Nov., 234 p., 6 pi. 

(22) HerkE, S. 

i913. contributions on nitrogen fixation and nutrition of bacillus 
radicicola and on bacterial tests op nitragin and azotogen. 
(Abstract.) In Exp. Sta. Rec, v. 29, no. 8, p. 733. (Original article 
in Kiserlet. Kozlem., v. 16, no. 3, p. 311-322, 1913. Not seen.) 

(23) Hills, T. L. 

the influence of nitrates on the growth of azotobacter. un- 
published. Offered for publication in the Ann. Rept. of the Pa. Agr. 
Exp. Sta. 

(24) HiLTNER, L. 

1900. UEBER DIE URSACHEN, WELCHE DIE GROSSE, ZAHL, STELLUNG UND 
WIRKUNG DER WURZELKNOLLCHEN DER LEGUMINOSEN BEDINGEN. In 

Arb. K. Gsndhtsamt., Biol. Abt., Bd. i, Heft 2, p. 177-222, pi. 3. 

(25) Immendorff, H. 

1892. BEiTRAGE ZUR LOSUNG DER " STiCKSToFFRAGE. " In Landw. Jahrb. 
Bd. 21, p. 281-339. 

(26) JosT, Ludwig. 

1907. LECTURES ON PLANT PHYSIOLOGY. Translated by R. J. H. Gibson. 
464 p., 172 fig. Oxford. Bibliography at the end of each lecture. 

(27) KaysER, E. 

1910. INFLUENCE DES nitrates SUR LES FERMENTS ALCOOLIQUES. /wCompt. 
Rend. Acad. Sci. [Paris], t. 151, no. 19, p. 816-817. 

(28) Kossowicz, Alexander. 

1914. UBER DAS VERHALTEN VON HEFEN UND SCHIMMELPILZEN ZU NITRATEN. 
In Biochem. Ztschr., Bd. 67, Heft 4/5, p. 400-419. 

(29) Laurent, Etnile. 

189I. RECHERCHES SUR LES NODOSIT^S RADICALES DES L^GUMINEUSES. In 

Ann. Inst. Pasteur, annee 5, p. 105-139, 3 fig. 

(30) Lewis, L. L., and Nicholson, J. F. 

1905. SOIL INOCULATION. TUBERCLE-FORMING BACTERIA OF LEGUMES. Okla. 
Agr. Exp. Sta. Bui. 68, 30 p., 8 fig. 

(31) Marchal, Emile 

I9OI. INFLUENCE DES SELS MIN^RAUX NUTRITIFS SUR LA PRODUCTION DES 

NODOSiT^S CHEZ LE POis. In Compt. Rend. Acad. Sci. [Paris], 
t. 133, no. 24, p. 1032-1033. 

(32) Maz6, P. 

1897. FIXATION DE L 'azote LIBRE PAR LE BACILLE DES NODOSIT^S DES 

LJfeGUMiNEUSES. In Ann. Inst. Pasteur, ann6e 11, no. i, p. 44-54- 

1898. LES MICROBES DES NODOSIT^S DES l6guminEUSES. In Ann. Inst. 

Pasteur, ann. 12, no. i, p. 1-25, i fig. 
(34) Meyer, Arthur. 

1912. DIE ZELLE DER BAKTERiEN. 285 p., 34 fig-, i col. pi. Jena. Literatur, 
p. 267-282. 



230 Journal of Agricultural Research voI.xii.no. 4 

(35) Moore, G. T. 

1905. soil inoculation for legumes ; with reports upon the successful 

use of artificial cultures by practical farmers. u. s. dept. 
Agr. Bur. Plant Indus. Bui. 71, 72 p., 10 pi. 

(36) MUNTER, F. 

I916. tJBER DEN EINFLUSS ANORGANISCHER SALZE AUF DAS WACHSTUM DER 

ACTINOMYCETEN. III. In Centbl. Bakt. [etc.], Abt. 2, Bd. 44, No. 
24/25, p. 673-695, 9 fig. 

(37) NoBBE, F., and Richter, L. 

1902. UBER DEN EINFLUSS DES NITRATSTICKSTOFFS UND DER HUMUSSUBS- 
STANZEN AUF DEN IMPFUNGSERFOLG BEI LEGUMINOSEN. In Landw. 

Vers. Stat., Bd. 56, Heft 5/6, p. 441-448. 

(38) PPEFPER, W. 

1906. THE PHYSIOLOGY OP PLANTS, ed. 2. Translated by A. J. Ewart. v. 3. 

Oxford. 

(39) Prazmowski, Adam. 

1891. DIE wurzelknollchen dEr ErbsE: ii. TEil. die biologischE BEDEU- 

TUNG DER WURZELKNOLLCHEN. In Landw. Vers. Stat., Bd. 38, p. 
5-56. 

(40) PringshEim, Hans. 

1914. ZUR STICKSTOPPASSIMILATION IN GEGENWART VON SALPETER. In 

Centbl. Bakt. [etc.], Abt. 2, Bd. 40, no. 1/8, p. 21-23. 

(41) Prucha, M. J. 

1915. PHYSIOLOGICAL STUDIES OF BACILLUS RADICICOLA OP CANADA FIELD PEA. 

N. Y. Cornell Agr. Exp. Sta. Mem. 5, 83 p. Bibliography, p. 79-83. 

(42) RiTTER, G. 

1909. AMMONIAK UND NITRATE ALS STICKSTOFFQUELLE FUR SCHIMMELPILZE. 

In Ber. Deut. Bot. Gesell., Bd. 27, Heft 10, p. 582-588. 

(43) SackETT„W. G. 

1915. The PIGMENT OF azotobacter chroococcum. In Proc. 35th Ann. 
Meeting Soc. Prom. Agri. Sci., 1914, p. 80-88, 2 col. pi. 

(44) Stoklasa, Julius. 

1908. BEITRAG ZUR KENNTNIS DER CHEMISCHEN VORGANGE BEI DER ASSIMI- 
LATION DES ELEMENTAREN STICKSTOPPS DURCH AZOTOBACTER UND 

RADiOBACTER. In Centbl. Bakt. [etc.], Abt. 2, Bd. 21, no. 15/16, p. 
484-511. 

(45) Vines, S. H. 

1888. on the relation between the formation of tubercles on the 
roots of leguminosae and the presence op nitrogen in the 
SOIL. In Ann. Bot., v. 2, no. 7, p. 386-389. 

(46) V/ILSON, J. K. 

I915. PHYSIOLOGICAL STUDIES OF BACILLUS RADICICOLA OF SOY BEAN. (Ab- 
stract.) In Science, n.s., v. 41, no. 1048, p. 180. 

(47) WOHLTMANN, Ferdinand, and bergen^, 

1902. DIE KNOLLCHEN-BAKTERIEN IN IHRER ABHANGIGKEIT VON BODEN UND 

dungung. /?^ Jour. Landw., Bd. 50, Heft 4, p. 377-395. 

(48) Woods, C. D. 

1892. the acquisition of atmospheric nitrogen by growing plants. in 

Conn. Storrs Agr. Exp. Sta., 4th Ann. Rpt., 1891, p. 17-28. 

(49) ZiPPEL, Hugo. 

I911. BEITRAGE ZUR MORPHOLOGIE UND BIOLOGIE DER KNOLLCHENBAKTERIEN 

DER LEGUMINOSEN. In Centbl. Bakt. [etc.], Abt. 2, Bd. 32, No. 
3/5> P- 97-137- Literature, p. 136-137. 



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